Nucleic acids encoding BCL2-associated athanogen 3 (BAG3) for gene therapy

Abstract

The present invention relates to recombinant nucleic acids and gene therapy vectors comprising nucleic acids encoding Bcl2-associated athrenogen 3 (BAG3) polypeptides, and variants thereof, for use in treating heart-related diseases and disorders, including those associated with BAG3 deficiency or dysfunction, such as dilated cardiomyopathy (DCM), and those not associated with BAG3 deficiency or dysfunction, such as heart failure unrelated to BAG3 expression.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 484,630, filed February 13, 2023, and U.S. Provisional Patent Application No. 63 / 501,898, filed May 12, 2023, the entire contents of each of which are incorporated herein by reference, including all text, tables, and drawings.

[0002] (Reference to sequence listing) This application has been filed electronically and includes an electronically submitted Sequence Listing. The Sequence Listing is entitled "24-0147-WO_SequenceListing.xml," was created on February 13, 2024, and is 67,384 bytes in size. The Sequence Listing contained in this .xml file is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Bcl2-associated athanogene 3 (BAG3) is involved in diverse cellular functions, including but not limited to excitation-contraction coupling, maintenance of sarcomere integrity, and regulation of autophagy (Kirk et al., J. Clin. Invest. 131(16):e149415(2021)). BAG3 possesses four protein-protein binding domains and acts through binding interactions with various other proteins (Qu et al., J. Am. Heart Assoc. 11(23):e027373(2022)). The various functions of BAG3 are thought to be protective in cardiomyocytes and include regulation of apoptosis, proteostasis, mitochondrial stability, myocardial contraction, and cardiac rhythm (Liu et al., Heart Fail. Rev. 26(1):183-94(2021)).

[0004] Mutations in BAG3 are associated with dilated cardiomyopathy (DCM) in humans (Myers et al., JAMA Cardiol. 3(10):929-38(2018)). BAG3-associated DCM (BAG3 DCM) is an autosomal dominant primary DCM. It is not associated with other systemic complications (asymptomatic). Patients with BAG3 DCM exhibit severely reduced cardiac function and moderate to severe symptoms of heart failure. BAG3 DCM is characterized by early presentation of heart failure (80% of genotype-positive patients older than 40 years exhibit DCM), a high incidence of heart failure in genotype-positive, phenotype-negative patients (26.1% at approximately 2-year follow-up), and a poor prognosis in the presence of heart failure symptoms, as evidenced by a 5.1% annual rate of death or major cardiovascular events (LVAD implantation, heart transplantation, SCD, or equivalent). Currently, patients with hereditary DCM lack precision medicine to address the underlying pathophysiology, and therapy remains undifferentiated from guideline-directed medical therapy (GDMT) for heart failure with reduced ejection fraction (HFrEF). The implementation of GDMT and device therapy for heart failure in DCM patients has improved overall prognosis. However, DCM patients still face a high unmet medical need. BAG3 DCM is a rare clinical indication with an estimated genetic prevalence of approximately 1% of all DCM patients that, based on the literature, could benefit from gene therapy treatment.

[0005] In addition to DCM and cardiac disease in general, BAG3 is a multifunctional protein involved in cellular stress responses through its involvement in several regulatory pathways that control cellular homeostatic responses in physiological and pathological conditions. Therefore, there is a need for treatment of any disease or disorder mediated by or associated with decreased BAG3 expression and / or activity, and moderate increases in BAG3 protein expression can lead to alleviation of the disease phenotype. Provided herein are tools and methods that function in human cells for the precise administration of BAG3. The compositions and methods provided herein modulate BAG3 levels, target disease, and can serve as the basis for curative therapies for many previously untreatable diseases and / or disorders, including, but not limited to, dilated cardiomyopathy. Summary of the Invention

[0006] The present disclosure provides gene therapy methods that deliver BAG3 protein expression to a desired target tissue (e.g., cardiac tissue). In some embodiments, the therapy can be used for the treatment of a heart-related disease or disorder, including, for example, a heart-related disease or disorder associated with a deficiency or dysfunction of BAG3. It is also intended to treat a heart-related disease or disorder unrelated to a deficiency or dysfunction of BAG3. Examples of heart-related diseases or disorders associated with a deficiency or dysfunction of BAG3 include, for example, BAG3-associated dilated cardiomyopathy (DCM) and BAG3-associated heart failure. Examples of heart-related diseases or disorders unrelated to a deficiency or dysfunction of BAG3 include, for example, heart failure unrelated to BAG3 expression.

[0007] In one aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof.

[0008] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof.

[0009] In another aspect, the disclosure provides an rAAV vector comprising, in 5' to 3' order: (a) at least one 5' ITR sequence; (b) at least one cardiac promoter; (c) at least one intron; (d) at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, operably linked to the at least one cardiac promoter; (e) at least one transcription termination sequence; (f) at least one stuffer or filler sequence; and (g) at least one 3' ITR sequence.

[0010] In another aspect, the disclosure provides an rAAV vector plasmid comprising, in 5' to 3' order: (a) at least one left spacer sequence; (b) at least one 5' ITR sequence; (c) at least one cardiac promoter; (d) at least one intron; (e) at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, operably linked to the at least one cardiac promoter; (f) at least one transcription termination sequence; (g) at least one stuffer or filler sequence; (h) at least one 3' ITR sequence; and (i) at least one right spacer sequence.

[0011] In another aspect, the disclosure provides a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, and at least one pharmaceutically acceptable salt.

[0012] In another aspect, the disclosure provides a method for treating a heart-related disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of (a) a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or (b) an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or (c) an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, and at least one pharmaceutically acceptable salt.

[0013] In another aspect, the disclosure provides a method for reducing the frequency or severity of at least one symptom associated with a heart-related disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition comprising: (a) a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof; or (b) an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof; or (c) a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof; and at least one pharmaceutically acceptable salt, in an amount effective to reduce the frequency or severity of the at least one symptom.

[0014] In another aspect, the disclosure provides the use of a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0015] In another aspect, the disclosure provides use of an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0016] In another aspect, the disclosure provides use of a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, or an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof, and at least one pharmaceutically acceptable salt, in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0017] In another aspect, the present disclosure provides a plasmid comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof. The present disclosure also provides a host cell for producing an rAAV vector comprising such a plasmid.

[0018] In another aspect, the present disclosure provides a plasmid comprising an rAAV vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide or a variant thereof. The present disclosure also provides a host cell for producing the rAAV vector comprising such a plasmid.

[0019] In another aspect, the present disclosure provides a method of making an rAAV vector, the method comprising: (a) incubating a host cell for rAAV vector production under conditions sufficient to allow production of the rAAV vector; and (b) purifying the rAAV vector produced thereby. The present disclosure also provides an rAAV vector produced by such a method.

[0020] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims, which should be noted that the claims are defined by the recitation therein, rather than by the specific discussion of the features and advantages described herein. [Brief explanation of the drawings]

[0021] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles and operation of the present disclosure. [Figure 1] 1 shows an exemplary efficacy evaluation of Compound A (multiplicity of infection, MOI: 3E4, 1E5, 3E5 vg / cell) in HiPSC-CMs based on detection of transgene mRNA (ddPCR) and total BAG3 protein (Wes™, an automated capillary-based immunoassay). [Figure 2] Figure 1 shows an exemplary efficacy evaluation of Compound A in HiPSC-CMs based on knockdown of endogenous BAG3 using siRNA, which leads to HSPB8 destabilization. Cells were then transduced with AAV (MOI: 1E5, 3E5 vg / cell) to drive BAG3 expression and result in HSPB8 stabilization. [Figure 3]Figure 3 shows exemplary biodistribution and expression assessment of human BAG3 in wild-type mice (C57BL / 6J) after administration of Compound A at 3 and 8 weeks post-dose. Dose: 3E13vg / kg (n: 12, male), control (n: 12). Note: Six mice were sacrificed at 3 weeks post-dose, and six mice were sacrificed at 8 weeks post-dose. Figure 3A shows exemplary longitudinal body weight measurements for the two groups. Figure 3B shows exemplary viral genome biodistribution in the heart at 3 and 8 weeks post-dose (left graph), and exemplary viral genome biodistribution in the heart, skeletal muscle (quadriceps), and liver at 8 weeks post-dose (right graph). Figure 3C shows exemplary transgene expression (mRNA) in the heart, skeletal muscle (quadriceps), and liver at 8 weeks post-dose. Normalized to mouse TBP gene expression levels. Figure 3D shows exemplary human-only BAG3 protein expression in the heart, skeletal muscle, and liver (left panel). Total BAG3 expression (mouse endogenous and human transgene) in the heart (right panel). Figure 3E shows exemplary ejection fraction and left ventricular end-diastolic volume determined using echocardiography at 4 and 7 weeks post-administration. Figure 3F shows exemplary spatial biodistribution of the transgene determined using immunohistochemistry (antibody specific for human-specific BAG3) at 3 and 8 weeks post-administration. [Figure 4]Exemplary characterization of a mouse disease model is shown. Figures 4A-4B show exemplary ejection fraction and left ventricular end-diastolic volume data measured using echocardiography in a cohort of mice with complete cardiac-specific knockdown of BAG3 (BAG3 cKO, n:10) and a cohort of wild-type controls (n:10). A mixed-effects model with an AR(1) covariance structure was performed to compare genotypes (BAG3 cKO vs. WT controls). # indicates statistical significance (p<0.05). Figure 4C shows exemplary longitudinal body weight measurements. Figures 4D-4E show exemplary BAG3 and HSPB8 protein expression levels quantified in the heart, liver, and skeletal muscle (quadriceps). # indicates statistical significance (p<0.05). Figures 4F-G show exemplary biomarkers of heart failure (Nppa, Nppb, Myh7 / Myh6 ratio) and fibrosis (Col1a1, Col1a2, Postn, Fn1, Timp1) quantified at the mRNA level (qRT-PCR) using RNA collected from cardiac tissue. # indicates statistical significance (p<0.05). Figure 4H shows an exemplary percentage of fibrotic area determined using picrosirius red staining. [Figure 5]Figure 5 shows an exemplary in vivo dose-response study to determine the effective dose and transgene expression associated with efficacy in a mouse model of disease. cKO control mice (n:20), Compound A 1E13vg / kg (n:15), Compound A 3E13vg / kg (n:14), Compound A 9E13vg / kg (n:14), and wild-type control (n:20). Figures 5A-5B show exemplary ejection fraction (EF, %) and left ventricular end diastolic volume (LVEDV, uL) data determined using echocardiography at 4, 8, and 12 weeks post-dose. A longitudinal mixed-effects model was performed to examine the % change from baseline in EF and LVEDV over the course of the study, controlling for gender. # indicates statistical significance (p<0.05). Figure 5C shows exemplary longitudinal body weight measurements (males). A longitudinal mixed-effects model with an AR(1) covariance structure was performed to compare body weight over the course of the study. Figure 5D shows exemplary tissue-specific biodistribution of Compound A, as determined by measuring viral genomes (ddPCR) in heart, liver, and skeletal muscle tissues collected at necropsy. Figure 5E shows exemplary tissue-specific expression of Compound A, as determined by measuring mRNA expression (RT-ddPCR) in heart, liver, and skeletal muscle tissues collected at necropsy. Figure 5F shows exemplary quantification of BAG3 protein expression in the heart (mouse endogenous + human transgene, Simple Western). Figure 5G shows exemplary quantification of HESPB8 protein expression in the heart (Simple Western). Figure 5H shows exemplary quantification of human BAG3 protein expression in the heart (LCMS). 5I-5J show exemplary spatial biodistribution expression analyses of transgene mRNA (in situ hybridization, ISH) and transgene protein (immunohistochemistry, IHC).Figure 5K shows exemplary biomarkers of heart failure (Nppa, Myh7 / Myh6 ratio) and fibrosis (Postn) measured (qRT-PCR) in cardiac tissue from wild-type control mice, BAG3 cKO control mice, or BAG3 cKO mice treated with 3E13vg / kg or 9E13vg / kg of Compound A. Statistical significance was determined using one-way ANOVA. * indicates statistical significance (p<0.05). [Figure 6A] Figure 6 shows exemplary biodistribution and expression assessment of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) after administration of Compound A. Doses: 4E13vg / kg (n:2, males and females), 1.3E14vg / kg (n:2, males and females), vehicle control (n:2, males and females). Note: The female animal administered the 1.3E14vg / kg dose was determined to seroconvert prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figure 6A shows exemplary biodistribution of the viral genome in three cardiac regions (R11, R12, R13), liver (right median lobe), dorsal root ganglia (DRG, thoracic), spinal cord (thoracic), skeletal muscle (gastrocnemius), and testis or ovary. [Figure 6B] Figure 6B-6C show exemplary biodistribution and expression assessments of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) following administration of Compound A. Doses: 4E13vg / kg (n: 2, males and females), 1.3E14vg / kg (n: 2, males and females), vehicle control (n: 2, males and females). Note: It was determined that the female animal administered the 1.3E14vg / kg dose seroconverted prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figures 6B-6C show exemplary transgene mRNA expression levels normalized to endogenous HPRT or endogenous BAG3 gene expression levels measured in three cardiac regions (R11, R12, R13), liver (right median lobe), dorsal root ganglion (DRG, thoracic), spinal cord (thoracic), and skeletal muscle (gastrocnemius). [Figure 6C]Figure 6B-6C show exemplary biodistribution and expression assessments of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) following administration of Compound A. Doses: 4E13vg / kg (n: 2, males and females), 1.3E14vg / kg (n: 2, males and females), vehicle control (n: 2, males and females). Note: It was determined that the female animal administered the 1.3E14vg / kg dose seroconverted prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figures 6B-6C show exemplary transgene mRNA expression levels normalized to endogenous HPRT or endogenous BAG3 gene expression levels measured in three cardiac regions (R11, R12, R13), liver (right median lobe), dorsal root ganglion (DRG, thoracic), spinal cord (thoracic), and skeletal muscle (gastrocnemius). [Figure 6D] Figure 6 shows an exemplary biodistribution and expression assessment of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) after administration of Compound A. Doses: 4E13vg / kg (n:2, males and females), 1.3E14vg / kg (n:2, males and females), vehicle control (n:2, males and females). Note: It was determined that the female animal administered the 1.3E14vg / kg dose seroconverted prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figure 6D shows an exemplary spatial cardiac biodistribution of transgene mRNA (percentage of positive cardiomyocytes) as determined by in situ hybridization (ISH, RNAScope). [Figure 6E]

[0023] Figure 1 shows an exemplary biodistribution and expression assessment of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) following administration of Compound A. Doses: 4E13vg / kg (n:2, males and females), 1.3E14vg / kg (n:2, males and females), vehicle control (n:2, males and females). Note: It was determined that the female animal administered the 1.3E14vg / kg dose seroconverted prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figures 6E-6F show exemplary BAG3 protein expression levels in the heart measured using liquid chromatography-mass spectrometry (LCMS) in three cardiac regions (R11, R12, R13), liver (right median lobe), dorsal root ganglion (DRG, thoracic), spinal cord (thoracic), and skeletal muscle (gastrocnemius). Quantification of total BAG3 was determined using a peptide conserved in both human and cynomolgus monkey BAG3. Quantification of human BAG3 was determined using a peptide specific to the human protein. [Figure 6F]

[0023] Figure 1 shows an exemplary biodistribution and expression assessment of human BAG3 in non-human primates (NHPs, cynomolgus monkeys) following administration of Compound A. Doses: 4E13vg / kg (n:2, males and females), 1.3E14vg / kg (n:2, males and females), vehicle control (n:2, males and females). Note: It was determined that the female animal administered the 1.3E14vg / kg dose seroconverted prior to administration, resulting in very low biodistribution and transgene expression. Data from this animal was excluded from the panel. Figures 6E-6F show exemplary BAG3 protein expression levels in the heart measured using liquid chromatography-mass spectrometry (LCMS) in three cardiac regions (R11, R12, R13), liver (right median lobe), dorsal root ganglion (DRG, thoracic), spinal cord (thoracic), and skeletal muscle (gastrocnemius). Quantification of total BAG3 was determined using a peptide conserved in both human and cynomolgus monkey BAG3. Quantification of human BAG3 was determined using a peptide specific to the human protein. [Figure 7] 1 shows exemplary LCMS data demonstrating a dose-dependent increase in human BAG3 protein localized in purified cardiac sarcomeres from cynomolgus monkeys treated with Compound A. [Figure 8] 1 shows exemplary immunohistochemistry data for human BAG3 protein in the hearts of individual WT mice treated with Compound A at 3 and 8 weeks post-treatment. [Figure 9] Exemplary in situ hybridization data for human BAG3 mRNA, shown as brown granules, in the heart, liver, and DRG (from left to right) of an individual WT mouse treated with Compound A at 8 weeks post-dose are shown. Skeletal muscle (not shown) was negative. [Figure 10] 1 shows exemplary H&E, immunohistochemistry, and in situ hybridization for human BAG3 protein and mRNA in the hearts of cKO mice treated with Compound A. [Figure 11A] Exemplary liver enzyme (ALT) elevations at ≧4E13vg / kg (FIG. 11A) are shown. [Figure 11B] Cytokine (TNF) responses at 1.3E14 vg / kg (FIG. 11B) are shown. [Figure 12] 1 shows an exemplary in situ hybridization for human BAG3 RNA in the heart of a cynomolgus monkey treated with Compound A. [Figure 13] Figure 13 shows an exemplary determination of the target therapeutic level of BAG3 expression. Changes in ejection fraction in BAG3-cKO mice treated with Compound A as a function of BAG3 protein (Figure 13A) and ISH-based BAG3-mRNA cardiomyocyte coverage (Figure 13B). Circles indicate observations for individual animals. Changes in ejection fraction in BAG3-cKO mice treated with Compound A as a boxplot of BAG3 expression minus BAG3 protein (Figure 13C) and ISH-based BAG3-mRNA cardiomyocyte coverage (Figure 13D). Mice with >20% protein expression or ISH+ cardiomyocyte coverage had better ejection fraction changes than mice with <20% expression but comparable to mice with >40% expression, suggesting saturation of cardiac function responses above 20% BAG3 protein / ISH-based expression. Group mean comparisons were performed using unpaired two-sample t-tests. The N for each boxed expression group is indicated below the respective boxplot. **=p<0.01 and ns=p>0.05. [Figure 14]Figure 14A shows an exemplary determination of the human effective dose for Compound A. Figure 14A shows the exemplary dose-dependence of cardiac transduction of Compound A (blue triangles) measured as VCN / dge in cynomolgus monkeys, consistent with the QSP model prediction (black line) based on historical data. Previous literature / in-house data on AAV9 cardiac transduction are also shown for comparison. Note that the model prediction that the human dose-transduction relationship was similar in cynomolgus monkeys and humans was consistent with reported clinical data for AAV9 (red symbols). Figure 14B shows an exemplary dose-dependence of cardiomyocyte coverage based on Compound A hBAG3 ISH in cynomolgus monkeys. Target levels of hBAG3 tissue coverage were achieved above 4E13 vg / kg in monkeys, and a similar dose response is expected in humans. Circles indicate measurements for individual heart sections from monkey studies (8 heart sections / monkey; n=2 at 4E13 vg / kg, n=1 at 1.3E14 vg / kg). Points and error bars indicate the mean and standard deviation. Figure 14C shows an exemplary dose-dependence of Compound A hBAG3 protein levels in cynomolgus monkeys. The target level of hBAG3 protein expression (22 ng BAG3 / mg protein; dashed line) was exceeded in monkeys at 1.3E14 vg / kg, but is predicted to be achieved in humans at a dose of 1E14 vg / kg. Lines and circles represent model predictions and observed data for individual monkeys, respectively. Points and error bars indicate the mean and standard deviation (3 heart slices / monkey; n=2 at 4E13 vg / kg, n=1 at 1.3E14 vg / kg). [Figure 15]Figure 15 shows exemplary data from an in vivo dose-response study to determine the effective dose and transgene expression relative to survival and efficacy in a mouse model of disease: cHET-naive control mice (Group 1, n:14), cHET mice treated with 3E13vg / Kg of Compound A (Group 2, n:13), cHET mice treated with 1E14vg / Kg of Compound A (Group 3, n:14), and cWT-naive control mice. Figure 15A shows the ejection fraction (EF, %), Figure 15B shows the left ventricular end-diastolic volume (LVEDV, uL), and Figure 15C shows the left ventricular end-systolic volume (LVESV, uL), all determined using echocardiography at baseline (2 weeks before treatment) and 3, 8, and 13 weeks after treatment. [Figure 16] Figure 16A shows exemplary mouse survival after treatment: untreated control cHET mice (group 1, n:14), cHET mice treated with 3E13vg / Kg of Compound A (group 2, n:14), cHET mice treated with 1E14vg / Kg of Compound A (group 3, n:14), and untreated control cWT mice (group 4, n:15). Kaplan-Meier survival analysis between groups and paired log-rank tests were used to compare survival rates among the three groups. There were no significant differences in survival among the treatment groups. Figure 16B shows exemplary longitudinal body weight measurements collected at baseline (2 weeks before treatment) and up to 15 weeks after treatment. [Figure 17]Figure 17A shows exemplary AAV biodistribution, transgene mRNA expression, BAG3 protein, and HSPB8 protein expression in the heart. Figure 17A shows exemplary tissue-specific biodistribution (heart) determined by measuring viral genomes (ddPCR) in heart tissue collected at necropsy from cHET mice treated with 3E13 vg / kg of Compound A (Group 2) and 1E14 vg / kg of Compound A (Group 3). Two control cohorts of mice were included: untreated cHET mice (Group 1) and untreated cWT mice (Group 4). Figure 17B shows exemplary transgene expression determined by measuring mRNA expression (ddPCR) in heart tissue collected at necropsy from the same cohort of mice. Figure 17C shows exemplary BAG3 protein expression determined by measuring its levels in heart tissue collected at necropsy from the same cohort of mice. Figure 17D shows exemplary HSPB8 protein expression levels determined by measuring its levels in cardiac tissue collected at autopsy from the same cohort of mice. Statistical analysis was performed using one-way ANOVA with multiple comparisons (Tukey's test). (ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, *** = p ≤ 0.0001). [Figure 18] Figures 18A-18E show exemplary expression of fibrosis-related biomarkers in the hearts of cHET mice treated with 3E13 vg / kg of Compound A (Group 2) and 1E14 vg / kg of Compound A (Group 3). Two control cohorts of mice were included: untreated cHET mice (Group 1) and untreated cWT mice (Group 4). Figures 18F-18H show exemplary expression of heart failure-related biomarkers in the hearts of cHET mice treated with 3E13 vg / kg of Compound A (Group 2) and 1E14 vg / kg of Compound A (Group 3). Two control cohorts of mice were included: untreated cHET mice (Group 1) and untreated cWT mice (Group 4). Statistical analysis was performed using one-way ANOVA with multiple comparisons (Tukey's test). (ns=p>0.05, *=p≦0.05, **=p≦0.01, ***=p≦0.001, ***=p≦0.0001). [Figure 19]Figure 19A shows exemplary mouse survival after treatment: untreated control Bag3 cKO mice (group 1, n:13), Bag3 cKO mice treated with 3E13 vg / kg of Compound A (group 2, n:16), and Bag3 cKO mice treated with 1E14 vg / kg of Compound A (group 3, n:15). Kaplan-Meier survival analysis and paired log-rank tests were used to compare survival rates among the three groups. Group 1 had a significantly lower survival rate compared with groups 2 and 3. Figure 19B shows exemplary longitudinal body weight measurements (males and females) collected at baseline (2 weeks before treatment) and up to 20 weeks after treatment. [Figure 20] Figure 20 shows an exemplary in vivo dose-response study to determine the effective dose and transgene expression associated with survival and efficacy in a mouse model of disease. Figure 20A shows exemplary ejection fraction (EF, %) data, Figure 20B shows exemplary left ventricular end-diastolic volume (LVEDV, uL) data, and Figure 20C shows exemplary left ventricular end-systolic volume (LVESV, uL) data, determined using echocardiography for untreated control Bag3 cKO mice (Group 1, n:13), Bag3 cKO mice treated with 3E13vg / Kg of Compound A (Group 2, n:16), and Bag3 cKO mice treated with 1E14vg / Kg of Compound A (Group 3, n:15). A longitudinal mixed-effects model with an AR(1) covariance structure was performed to compare each variable between groups over time, controlling for gender. Symbols represent means and error bars represent standard deviations (ns=p>0.05, *=p≦0.05, **=p≦0.01, ***=p≦0.001, ***=p≦0.0001). [Figure 21]Figure 21A shows exemplary tissue-specific biodistribution (heart) determined by measuring viral genomes (ddPCR) in cardiac tissue collected at necropsy from untreated control Bag3 cKO mice (Group 1), Bag3 cKO mice treated with 3E13 vg / Kg of Compound A (Group 2), and Bag3 cKO mice treated with 1E14 vg / Kg of Compound A (Group 3). Figure 21B shows exemplary transgene expression determined by measuring mRNA expression (ddPCR) in cardiac tissue collected at necropsy. Figure 21C shows exemplary transgene BAG3 protein expression determined by measuring its levels in cardiac tissue collected at necropsy. BAG3 protein expression was normalized to BAG3 levels detected in the hearts of wild-type mice. Statistical analysis was performed using one-way ANOVA with multiple comparisons (Tukey's test). (ns=p>0.05, *=p≦0.05, **=p≦0.01, ***=p≦0.001, ***=p≦0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0022] The following discussion is directed to various embodiments. This disclosure is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. While one or more of these embodiments may be preferred, the disclosed embodiments should not be construed or otherwise used as limiting the scope of the disclosure, including the claims. Furthermore, those skilled in the art will appreciate that the following description is broad in scope and that the discussion of any embodiment is intended only as an example of that embodiment and is not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0023] The present disclosure provides gene therapy methods that deliver BAG3 protein expression to a desired target tissue (e.g., cardiac tissue). In some embodiments, the therapy can be used for the treatment of a heart-related disease or disorder, including, for example, a heart-related disease or disorder associated with a deficiency or dysfunction of BAG3. It is also intended to treat a heart-related disease or disorder unrelated to a deficiency or dysfunction of BAG3. Examples of heart-related diseases or disorders associated with a deficiency or dysfunction of BAG3 include, for example, BAG3-associated dilated cardiomyopathy (DCM) and BAG3-associated heart failure. Examples of heart-related diseases or disorders unrelated to a deficiency or dysfunction of BAG3 include, for example, heart failure unrelated to BAG3 expression.

[0024] The extent and level of BAG3 protein expression required to halt the deterioration of cardiac structure and function were evaluated in a BAG3 cardiac knockout (cKO) mouse model. These biodistribution targets were demonstrated in nonhuman primates (NHPs) at clinically feasible dose levels with acceptable safety. Toxicology evaluations in mice (WT and cKO) and NHPs provided an adequate safety profile. Modeling predicted clinically effective dose levels of 3E13vg / kg to 1E14vg / kg, and doses up to 1.3E14vg / kg were tested in NHPs.

[0025] Specific Definitions As used herein, the terms "about" and "approximately" refer to an amount that is ±10% of the recited value, optionally ±5% of the recited value, or more optionally ±2% of the recited value. The terms "about" and "approximately" are also used to provide literal support for the exact number that it precedes, as well as a number that is close to or approximately the number that the term precedes. When determining whether a number is close to or approximately a specifically recited number, the unrecited close or approximate number may be a number that, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0026] As used herein, "adeno-associated viral vector" refers to an adeno-associated virus (AAV) that contains a naturally occurring or non-naturally occurring AAV capsid that encapsidates the vector. Adeno-associated viral vectors may be abbreviated as "AAV vectors" and may be referred to by synonyms such as "recombinant AAV vectors," "rAAV vectors," "rAAV," or simply "vectors," depending on the context.

[0027] As used herein, "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. The vectors (e.g., recombinant AAV vectors) described herein can be administered by any method known to those skilled in the art. Suitable methods for administering vectors can include, for example, infusion or injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal, and subcutaneous), instillation preparations, and the like. The method of administering a vector can include subcutaneous administration. The vectors prepared as described herein can be administered in various forms depending on the disorder being treated and the age, condition, and weight of the subject, as is known in the art. The preparation can be administered prophylactically, i.e., administered to reduce the likelihood of developing a disease or condition.

[0028] As used herein, the term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 10 nucleotides of a 21-nucleotide nucleic acid molecule" means that a range of 10 to 21 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides, has the specified property. When "at least" is present before a series or range, it is understood that "at least" can modify each of the numbers in the series or range.

[0029] As used herein, the term "cardiac promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced substantially in a living cell when the cell is a cell of cardiac tissue.

[0030] As used herein, the terms "complementary DNA" or "cDNA" are interchangeable and refer to a nucleic acid sequence that is the DNA equivalent of an mRNA sequence (i.e., has uridines substituted for thymidines). Generally, the terms cDNA and mRNA can be used interchangeably with respect to a particular gene (e.g., the BAG3 gene), such that one skilled in the art will understand that the cDNA sequence is the same as the mRNA sequence, except that uridines are read as thymidines.

[0031] As used herein, the term "disease" refers to the interruption, cessation, or impairment of a bodily function, system, or organ. Diseases or disorders of interest include those that may benefit from treatment with a vector such as those described herein that delivers BAG3 expression to target tissue. For example, diseases or disorders of interest include those that may benefit from treatment with a rAAV vector described herein that delivers BAG3 expression to cardiomyocytes, such as by the therapeutic methods described herein. Non-limiting examples of diseases or disorders mediated by or associated with BAG3 protein expression that can be treated using the compositions and methods described herein include, for example, BAG3 DCM and BAG3-associated heart failure.

[0032] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" of an agent (e.g., an rAAV vector described herein) that delivers human BAG3 protein expression to cardiomyocytes (e.g., in a subject) refer to an amount sufficient to effect a beneficial or desired result, including a clinical result, when administered to a subject, including a human; therefore, an "effective amount" or its equivalents will depend on the context in which it is applied. For example, in the context of treating a disease associated with BAG3 protein expression, it is the amount of agent that delivers sufficient BAG3 protein expression to achieve a therapeutic response or beneficial or desired result compared to the response obtained without administration of the agent.

[0033] As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a formulation or composition to, for example, provide or contribute to a desired consistency or stabilizing effect.

[0034] As used herein, "expression cassette" means a nucleotide sequence comprising a transgene operably linked to a promoter, regulatory region, or regulatory element to control the initiation and termination of transcription of the transgene from DNA to RNA.

[0035] As used herein, the terms "formulation," "pharmaceutical formulation," and "pharmaceutical composition," as they relate to the vectors described herein, are intended to describe a vector (e.g., rAAV) in combination with one or more pharmaceutically acceptable excipients, including, for example, buffers, salts, cryoprotectants, and / or surfactants, optionally with a defined pH, and further optionally, where the formulation is manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. A "pharmaceutical formulation" or "pharmaceutical composition" is a preparation that is in a form such that the biological activity of the active ingredient is effective. Pharmaceutical formulations and compositions can be formulated, for example, for subcutaneous administration, intravenous administration (e.g., as a sterile solution in a solvent system that is free of particulate plugs and suitable for intravenous use), intrathecal injection, intraventricular injection, intraparenchymal injection, oral administration in a unit dosage form (e.g., a tablet, capsule, caplet, gel capsule, or syrup), or topical administration (e.g., as a cream, gel, lotion, or ointment, or any other pharmaceutically acceptable formulation).

[0036] As used herein, "heterologous nucleotide sequence" means a nucleotide sequence that is introduced into one organism (including a virus) from a different organism (including an organism). The sequence of a heterologous nucleotide sequence can be the same as that found in nature, or it can be a modified version thereof, or it can even be partially or completely synthetic.

[0037] As used herein, "host cell" means a cell in which a viral vector is produced. Producer cells and packaging cells are examples of host cells. Host cells, whether unicellular or multicellular, can be derived from mammals or insects, or other organisms.

[0038] As used herein, "percent sequence identity" with respect to a reference nucleic acid or polypeptide sequence is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in the reference nucleic acid or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent nucleic acid or amino acid sequence identity can be accomplished in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleotide or amino acid sequence A to a given nucleotide or amino acid sequence B, or relative to a given nucleotide or amino acid sequence B, is calculated as follows (alternatively, one can say that a given nucleotide or amino acid sequence, A, has a certain percent sequence identity to a given nucleotide or amino acid sequence B, or relative to a given nucleotide or amino acid sequence B): Multiply 100 by (fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic alignment of A and B, and where Y is the total number of nucleotides in B. It will be understood that if the length of nucleotide or amino acid sequence A is not equal to the length of nucleotide or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0039] As used herein, the term "purify," and its related terms "purified," "purified," and "isolated," when used in connection with the vectors described herein, or samples or preparations thereof, indicates a relative increase or improvement in purity compared to the starting material comprising the vector and / or compared to a previous intermediate purification step in some schemes of sequential purification steps intended to purify a biological product, and does not require a particular qualitative or quantitative purity, unless otherwise specified.

[0040] As used herein, "subject" refers to an organism to which a vector (e.g., rAAV) is administered for the purpose of preventing or treating a disease, disorder, or condition. Furthermore, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can be a human or animal that seeks or needs treatment, requests treatment, is undergoing treatment, will undergo treatment in the future, or is receiving care from a professional trained in a particular disease or condition.

[0041] As used herein, "target cell" means a cell that a vector (e.g., rAAV) is designed or intended to transduce, or a cell that has been experimentally observed to be transduced by the vector, whether in vitro or in vivo in a subject.

[0042] As used herein, "transduction" refers to the introduction of the genome of a vector (e.g., rAAV) into a target cell. Transduction is distinguished from infection, the latter term being used to refer to the introduction of a replication-competent viral genome into a cell.

[0043] As used herein, "transgene" means a nucleotide sequence that encodes at least one polypeptide and / or a nucleotide sequence that encodes at least one functional RNA molecule. A transgene may also be referred to by the synonym "gene of interest."

[0044] As used herein, the term "treating" refers to the act of providing care to a subject in need thereof, for example, through the administration of a therapeutic agent (e.g., an rAAV described herein) to the subject for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, a disorder) or to prevent or reduce the likelihood of the occurrence of a condition. In some embodiments, treatment comprises reducing the frequency or severity of at least one sign, symptom, or contributor to a condition (e.g., a disease, a disorder) experienced by the subject.

[0045] As used herein, "vector" refers to an AAV genome that contains a heterologous nucleotide sequence and that has been modified to render any AAV vector containing the vector replication-incompetent, e.g., by inactivating or deleting the endogenous AAV rep and / or cap genes.

[0046] BAG3 The BAG3 gene is located on chromosome 10q26.11 (MIM:603883) (nucleotide NCBI reference sequence: NM_004281.4; protein NCBI reference sequence: NP_004272.2) and is referred to as "BCL2-related athogen 3," "BAG co-chaperone 3," or "BAG family molecular chaperone regulator 3." BAG3 is a multifunctional protein involved in cellular stress responses through participation in several regulatory pathways that control cellular homeostatic responses under physiological and pathological conditions. BAG proteins compete with Hip for binding to the Hsc70 / Hsp70 ATPase domain, promoting substrate release. The BAG domains of BAG1, BAG2, and BAG3 specifically interact with the Hsc70 ATPase domain in vitro and in mammalian cells, inhibiting its chaperone activity in a Hip-inhibitory manner.

[0047] BAG3 mutations BAG3 mutations in humans are primarily nonsense, deletion, or frameshift mutations, all of which result in similar disease phenotypes. Cardiac BAG3 knockout (cKO) mice exhibit heart failure, dilation, and fibrosis. Delivery of BAG3 by adeno-associated virus (AAV) vectors stabilizes cardiac structure and function and reduces biomarkers of heart failure and fibrosis. Furthermore, BAG3 protein levels are reduced in failing human hearts (without BAG3 mutations), and AAV delivery of BAG3 protects WT mouse hearts from ischemia / reperfusion injury.

[0048] Mutations in BAG3 are associated with a rare autosomal dominant form of nonsyndromic dilated cardiomyopathy (DCM 1HH / BAG3; OMIM 613881), based on the literature, with an estimated genetic prevalence of approximately 1% of all DCM patients.

[0049] Patients with BAG3-associated DCM exhibit severely reduced cardiac function and moderate to severe symptoms of heart failure. Once diagnosed with BAG3-associated DCM, patients experience a high cardiac event rate of approximately 25-30% (e.g., death, LVAD, heart transplant, sustained VT, etc.), with a 5% mortality rate over a 4-5 year period (Dominguez et al., J. Am. Coll. Cardiol. 72(20):2471-81(2018)). BAG3 expression may be downregulated in heart failure in general and therefore may be a target for acquired heart failure.

[0050] Adeno-associated virus (AAV) The present disclosure provides vectors made from recombinantly modified adeno-associated viruses (AAV). AAV vectors can deliver genes, which may be under the control of transcriptional and other regulatory elements, to target cells via transduction. AAV vectors are useful in gene therapy for various diseases and disorders by providing functional copies of genes to target cells lacking or mutated endogenous versions.

[0051] AAV is a small, non-enveloped, non-pathogenic parvovirus that depends on certain other viruses to provide gene products known as helper factors essential for its replication, making it well suited to function as a recombinant vector. For example, adenovirus (AdV) can function as a helper virus in cells co-infected with adenovirus and AAV by providing specific adenoviral factors, such as E1A, E1B55K, E2A, and E4ORF6 proteins, as well as VA RNA. Other helper viruses, such as herpes simplex virus, have also been identified. Because AAV replication depends on accessory factors provided by other viruses, AAV is classified as a type of dependovirus. The AAV virion has two major structural features, termed the capsid and the genome. The capsid is an icosahedral protein shell that encloses and protects (encapsidates) the viral genome, which contains genes and other sequences necessary for viral replication within infected cells.

[0052] The AAV genome is a single strand of DNA containing two genes, rep and cap. For example, in AAV9, a naturally occurring AAV that infects humans and is biologically well characterized, the genome is approximately 4.7 kilobases in length. Alternative splicing of transcripts from two promoters allows the rep gene to produce four related multifunctional proteins called Reps (e.g., Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weights), which are involved in viral gene expression and genome replication and packaging. Alternative splicing of transcripts from a single promoter controlling a single cap gene produces three related structural proteins, VP1, VP2, and VP3, which self-assemble into a total of 60 proteins that form the viral icosahedral capsid in an approximately 1:1:10 ratio. VP1 is the longest of the three VP proteins and contains amino acids in its amino-terminal region that are not present in VP2, which in turn is longer than VP3 and contains amino acids in its amino-terminal region that are not present in VP3. In addition to containing the genome, the capsid protein mediates interaction through specific binding with receptors on the surface of target cells. Based on this, AAVs can be limited in their infectivity to specific animal species and even tissues within the same type of animal, a phenomenon called tropism. For example, some types of AAVs can preferentially infect liver cells (e.g., hepatocytes) over muscle or nerve cells.

[0053] In addition to the rep and cap genes, the intact AAV genome contains relatively short (145 nucleotide) sequence elements located at each of its 5' and 3' ends, called inverted terminal repeats (ITRs). The ITRs contain nested palindromic sequences that can self-anneal by Watson-Crick base pairing to form T-shaped or hairpin-shaped secondary structures. ITRs have been shown to have important functions required for the viral life cycle, including converting the single-stranded DNA genome to the double-stranded form required for gene expression and packaging the single-stranded AAV genome into capsid assembly by the Rep protein.

[0054] Numerous naturally occurring types of AAV have been discovered in different species. Previously, only six types of primate AAV were isolated from biological samples (AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6), and the first five of these had sufficient structural differences to be classified as distinct serotypes based on antibody cross-reactivity experiments. Later, two novel AAVs, designated AAV7 and AAV8, were discovered by PCR amplification of DNA from rhesus macaques using primers targeting highly conserved regions in the cap genes of previously discovered AAVs (Gao et al., Proc. Natl. Acad. Sci. USA 99(18):11854-59 (2002)). Subsequently, using a similar approach, numerous novel AAVs were cloned from human and nonhuman primate tissues, significantly expanding the scope of AAV capsid protein sequences (Gao et al., J. Virol. 78(12):6381-88 (2004)). Although many AAV capsid protein sequences are highly similar to each other or to previously identified AAVs and are often referred to as distinct AAV "serotypes," it is not necessarily expected that all will be immunologically distinguishable when such capsids are tested by antibody cross-reactivity. AAVs or AAV capsids that are serologically indistinguishable from a defined serotype but contain capsid proteins with different amino acid sequences are better referred to as variants of a known serotype. Many capsids made from naturally occurring and non-naturally occurring capsid proteins have proven useful for generating AAV gene therapy vectors.

[0055] After binding to one or more receptor molecules on the cell surface, AAV viral particles enter the cell via endocytosis. Upon reaching the low pH of the lysosome, the capsid protein undergoes a conformational change, allowing the capsid to escape into the cytosol and then be transported into the nucleus. Once inside the nucleus, the capsid disassembles, releasing the genome, and is acted upon by cellular DNA polymerase to synthesize a second DNA strand starting from the ITR at the 3' end, which acts as a primer after self-annealing. Expression of the rep and cap genes then begins, allowing the subsequent formation of new viral particles and their release from the cell.

[0056] AAV9 is a human AAV serotype with enhanced transduction efficiency in cardiac and skeletal muscle, liver and pancreatic tissue, and the eye compared with other serotypes. Like other AAVs, AAV9 can transduce non-dividing cells, including hepatocytes, which normally express factor IX (FIX). AAV9 can also cross the blood-brain barrier, and among all AAVs, it targets the central nervous system with high efficiency. Furthermore, although pre-existing antibodies to AAV have been shown to be detrimental to AAV gene delivery, the prevalence of antibodies to AAV9 is lower in humans than to other serotypes, such as AAV1 and AAV2, making this serotype a more attractive candidate for development as a gene delivery vector.

[0057] AAV vectors The relatively simple structure and life cycle of AAV, as well as the fact that AAV is known not to be pathogenic in humans, motivated researchers to explore whether AAV could be engineered and converted from a virus into a recombinant vector for gene therapy. Briefly, this was accomplished by cloning the entire AAV2 genome, including both ITRs, into a plasmid, moving the rep and cap genes to another plasmid, and replacing them with a heterologous gene expression cassette containing a promoter controlling the transgene-encoding protein. Thus, the only viral genomic sequences retained in the vector were the ITRs, because they perform essential functions in packaging and gene expression; without them, the AAV vector could neither be produced nor function to express a transgene after transduction of target cells. Finally, to avoid the need for coinfection with a helper virus, which is necessary for AAV virion replication, the genes for the so-called helper factors (e.g., for AdV, E1A helper factor, E1B55K helper factor, E2A helper factor, E4ORF6 helper factor, and VA RNA helper factor) were cloned into a third plasmid.

[0058] When the three plasmids (sometimes referred to as the transgene, rep / cap, and helper plasmids) were transfected together into mammalian host cells, the Rep and capsid proteins, as well as the helper virus factors, were expressed from their respective plasmids. These gene products then functioned within the host cells to replicate the vector from the plasmid on which they resided into single-stranded DNA, assemble capsids, and package the single-stranded genome into the capsids to form the vector. The vector could then be purified from the host cells. Because the rep and cap genes were present in trans on a different plasmid, outside of their usual context adjacent to the ITRs, they were not packaged into the vector. As a result, the AAV vectors produced in this manner were able to bind to and deliver the expression cassette within their genome into target cells, but they were unable to replicate and generate new vector particles.

[0059] If the vector functions as intended, after transduction, the expression cassette becomes transcriptionally active in the target cell, producing the gene product encoded by the transgene. AAV vectors are extremely versatile because they can be designed to contain various transgenes in various configurations under the control of various functional sequences and regulatory elements, and can be combined with various naturally occurring capsids and engineered capsids with various tropisms and other properties. Thus, it is possible to produce a large number of types of gene products, with some control over the type of cell to be transduced and the amount of gene product produced.

[0060] An AAV vector comprises a vector encapsidated by an AAV capsid. In some embodiments, the AAV vector comprises at least one AAV inverted terminal repeat (ITR) and a heterologous nucleotide sequence that has a desired function when present or expressed in a transduced target cell. In some embodiments, the heterologous nucleotide sequence is derived from a different type of virus or an entirely different type of organism, such as an animal, plant, protist, fungus, bacterium, archaea, or other type of organism. In some embodiments, the heterologous nucleotide sequence replaces part or all of the native AAV rep and / or cap genes such that the vector is unable to express functional Rep or VP proteins in the transduced target cell. In some embodiments, the entire sequence of the vector consists of the heterologous nucleotide sequence, except for the AAV inverted terminal repeats located at the ends of the genome.

[0061] The genome length of the AAV vectors of the present disclosure, including the ITRs, can be any suitable length, which typically, but not necessarily, does not exceed the average genome size packaging capacity of a particular AAV capsid, which may be selected in the design and production of a particular AAV vector. Thus, in some non-limiting embodiments, the genome length of the AAV vectors of the present disclosure, including the ITRs, is at least or about 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, It may be 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, or 5200 nucleotides (or base pairs when the genomic sequence is embodied in a plasmid for vector production), or any integer value between or range inclusive of any of the specifically recited values ​​above.

[0062] The length of the AAV plasmid-based vector of the present disclosure can be any suitable length. In some non-limiting embodiments, the length of the AAV plasmid-based vector of the present disclosure is at least or about 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 1000, 10100, 800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, or 12000 nucleotides, or any integer value between or inclusive of any of the specifically recited values ​​above.

[0063] AAV capsid protein The AAV vectors of the present disclosure can utilize any AAV capsid protein, whether naturally occurring, modified, or engineered, including those currently known or yet to be discovered or developed, that is suitable for transducing cells of a subject to express a BAG3 protein or a variant thereof from a vector transgene.

[0064] Numerous considerations and factors can guide the selection of capsid proteins (and the corresponding cap gene sequences used in their production) used in the design and production of AAV vectors. As described above, by specifically interacting with particular cell surface receptors, different AAV capsids can have different cell or tissue tropisms, which can be advantageous when it is desired to transduce certain tissues relative to other tissues. For example, to express a transgene product in the heart, such as cardiomyocytes, a vector can be designed and produced using a capsid with a stronger tropism for cardiomyocytes compared to, for example, neurons or hepatocytes. Conversely, to express a transgene product in the liver, a vector can be designed and produced using a capsid with a stronger tropism for hepatocytes compared to neurons, muscle, or other tissues.

[0065] Other factors may also be important. For example, it has been reported that some humans have high neutralizing antibody titers against certain capsids as a result of exposure to naturally occurring AAV, which may interfere with the ability of AAV vectors with the same or similar capsids to transduce target cells. Therefore, when designing vectors for gene therapy, the selection of a capsid may in some cases be guided by the capsid's immunogenicity and / or seroprevalence in the patient to be treated. Other considerations that may influence the selection of a capsid include manufacturability and stability during storage, and other relevant guiding factors are known in the art.

[0066] The AAV vectors of the present disclosure can use capsids made from capsid proteins derived from naturally occurring AAVs, as well as modified or engineered capsid proteins. For example, naturally occurring capsid proteins can be modified by inserting or deleting amino acids or peptides or by introducing amino acid substitutions in the VP1, VP2, and / or VP3 protein sequences with the intention of improving capsid function in some respect, such as tissue tropism, immunogenicity, stability, or manufacturability. Other examples include novel capsids with improved properties created by exchanging amino acids or domains from one known capsid to another (e.g., a mosaic or chimeric capsid), or by using DNA shuffling and directed evolution methods to discover capsid protein sequences with desired properties.

[0067] In some embodiments, AAV vectors of the disclosure can comprise capsids from known AAV serotypes and variants, as well as non-naturally occurring capsids, including, but not limited to, serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12), among others. In some embodiments, the capsid of an AAV vector of the disclosure comprises VP1, VP2, and / or VP3 AAV capsid proteins that are variants or derivatives of the known VP1, VP2, or VP3 AAV capsid proteins. In some embodiments, the amino acid sequence of such a variant or derivative AAV capsid protein is any known AAV capsid protein, including, but not limited to, the AAV capsid VP1, VP2, or VP3 protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or any other suitable AAV capsid, including, for example, the heart capsid discussed below. The amino acid sequence of the AAV capsid VP1, VP2, or VP3 protein sequence may be at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the amino acid sequence of the AAV capsid VP1, VP2, or VP3 protein sequence ofIn some other embodiments, the amino acid sequence of such variant or derivative AAV capsid proteins is a known AAV capsid VP1, VP2, or VP3 protein, including, but not limited to, the AAV capsid VP1, VP2, or VP3 protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or any other suitable AAV capsid, including, for example, the heart capsid discussed below. The amino acid sequence of the target polypeptide may differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids from the target amino acid sequence (whether due to amino acid deletions, insertions, or substitutions).

[0068] Cardiotropic AAV capsid The heart is an important target organ for BAG3 gene therapy. In some embodiments, the rAAV vector containing the BAG3 transgene contains a capsid protein known in the art as cardiac tropic. Non-limiting examples of cardiac tropic capsid proteins include one or more of AAV1, AAV4, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 capsid proteins. For example, AAV9 is known to transduce the heart in non-human primates (Pacak et al., Circ. Res. 99(4):e3-9(2006)). The cTNT cardiac promoter can be used to express the BAG3 transgene in transduced cardiomyocytes and restrict expression outside the heart (Prasad et al., Gene Ther. 18(1):43-52(2011)).

[0069] In some embodiments, the AAV vector comprises AAV9 capsid proteins, which may include VP1 protein (e.g., the amino acid sequence of SEQ ID NO: 1), VP2 protein (e.g., the amino acid sequence of SEQ ID NO: 2), and VP3 protein (e.g., the amino acid sequence of SEQ ID NO: 3), or functional subsequences, modifications, or variants thereof.

[0070] Expression cassette In some embodiments, the heterologous nucleotide sequence comprises or consists of an expression cassette or "payload" that includes a transgene operably linked to a promoter and, optionally, one or more enhancers, which act to control the initiation of transcription of the transgene from DNA to RNA, and a transcription termination element, such as a polyadenylation signal sequence, which acts to terminate transcription of the transgene into RNA. An AAV vector can contain two or more transgenes, either as part of a single transcription unit or each part of its own transcription unit. As described in later sections, the expression cassette can further include additional sequence elements designed to affect transcription, transcript stability, translation, or other functions.

[0071] AAV vectors are typically designed so that the structure of the expression cassette and the entire genome are limited by the packaging capacity of the capsid, such that the length of the transgene, together with all other elements in the genome required for vector function, such as transcriptional regulatory elements and ITRs, does not exceed approximately 4.7 kilobases for AAV9, although other types of capsids may have larger or smaller packaging limits. However, within size constraints, there is great flexibility in the selection of the transgene, ITRs, and other elements required for the vector to function for its intended purpose.

[0072] In some embodiments, the expression cassette of an rAAV of the present disclosure ranges in size from about 3.5 to about 4.7 kb, hi certain embodiments, the expression cassette of an rAAV of the present disclosure is about 4.2 kb in size.

[0073] BAG3 transgene In some embodiments, the AAV vectors of the present disclosure include vectors comprising an expression cassette containing a nucleotide coding sequence (transgene) for a BAG3 protein, or a variant thereof. In some embodiments, the BAG3 protein is identical to the 575 amino acid human BAG3 protein (NCBI Reference Sequence NP_004272.2 or SEQ ID NO: 10).

[0074] In yet other embodiments, the BAG3 protein may include any naturally occurring variant of the human BAG3 protein that does not contain pathogenic mutations, such as premature translation termination codons or amino acid substitutions, insertions, or deletions, that substantially impair BAG3 activity and / or protein stability. In still further embodiments, the BAG3 protein may include engineered human BAG3 protein variants, e.g., chimeric variants, that retain BAG3 activity, and variants with amino acid substitutions, insertions, or deletions designed to modulate BAG3 activity, add or remove glycosylation sites, add, remove, or alter internal cleavage sites or sites for other post-translational modifications, or alter other aspects of BAG3 structure or function.

[0075] In certain other embodiments, the BAG3 protein variant comprises at least one amino acid substitution mutation, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids, compared to the full-length wild-type human BAG3 protein amino acid sequence (such as that provided by SEQ ID NO: 10), altered from the wild-type counterpart. In some of these embodiments, the substitution mutation may be a conservative amino acid substitution, in which a normally occurring amino acid is replaced with another amino acid that has similar physicochemical and / or size characteristics and an R group. Alternatively, in other embodiments, the substitution mutation may be a non-conservative amino acid substitution, in which a normally occurring amino acid is replaced with another amino acid that has dissimilar physicochemical and / or size characteristics and an R group.

[0076] For use in the AAV vectors of the present disclosure, the nucleotide sequence encoding the BAG3 protein can be any nucleotide sequence capable of encoding the desired BAG3 protein in a cell type, such as a cardiomyocyte, that is desired to be transduced by the vector. In some embodiments, the nucleotide sequence encoding the BAG3 protein (i.e., the transgene) is the same as that present in a naturally occurring gene encoding BAG3 (i.e., the exons of such a gene), or is a DNA sequence corresponding to the mRNA sequence transcribed from such a gene. In some embodiments, when the BAG3 protein is a full-length wild-type human BAG3 protein, the encoding nucleotide sequence is SEQ ID NO:4.

[0077] In other embodiments, a nucleotide sequence encoding a BAG3 protein may differ at one or more nucleotide positions compared to a naturally occurring nucleotide sequence and, due to genetic code redundancy, may still encode a BAG3 protein identical to the naturally occurring gene sequence or a BAG3 protein variant encoded differently by the naturally occurring gene sequence, except for polypeptide differences relative to wild-type BAG3. In some embodiments, the nucleotide sequence encoding a BAG3 protein may be intentionally modified to affect its function in transduced cells, for example, to eliminate sequence motifs that can stimulate an innate immune response, to eliminate potential splice junctions, to eliminate alternative start codons, to increase the stability of the corresponding mRNA, and / or to increase the rate of translation of the mRNA into protein. In other embodiments, the nucleotide sequence encoding a BAG3 protein may be intron-free or may contain one or more introns that interrupt the coding sequence but are removed by the splicing machinery in the transduced cells to allow translation of the desired BAG3 protein.

[0078] In some embodiments of the AAV vectors of the present disclosure, the transgene encodes a protein sequence that is highly similar or identical to the protein sequence encoded by a particular nucleotide reference sequence, although the nucleotide sequences of the transgene and the reference sequence are not identical; rather, they share a particular percent identity, with the differences corresponding to positions within the codons that do not change the corresponding amino acid (i.e., are silent changes). For example, in some embodiments, the transgene comprises or consists of a nucleotide sequence encoding the same full-length BAG3 protein as set forth in SEQ ID NO: 10, wherein the nucleotide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the nucleotide sequence of SEQ ID NO: 4.

[0079] The percentage of nucleotide sequence identity between a reference sequence and a transgene can be determined by any method known in the art. For example, in some embodiments, a computer algorithm can be used to align and compare the nucleotide sequences (or the amino acid sequences encoded by them) of a reference sequence and a transgene over their entire lengths, and calculate the percent nucleotide sequence identity. An exemplary algorithm for globally aligning and comparing nucleotide sequences is the Needleman-Wunsch algorithm. However, in other embodiments, local alignment algorithms such as the BLAST algorithm can be used (Needleman et al., J. Mol. Biol., 48(3):443-53(1970); States et al., Methods, 3(1):66-70(1991); Pearson, Curr. Protoc. Bioinformatics, 43:3.5.1-3.5.9(2013)). In some embodiments, if one or the other of the reference sequence and the transgene sequence contains non-coding sequences, such as introns or stop codons, the non-coding sequences are ignored and only the protein-coding sequences in the reference sequence and the transgene sequence are aligned and compared. After an optimal global alignment between the reference sequence and the transgene is established, the percentage of identical nucleotides between the aligned sequences can be calculated.

[0080] As is known in the art, sequence comparison algorithms allow the user to define parameters, such as substitution scores and gap penalties, used to calculate alignment scores for the many possible alignments that can be created. The alignment with the highest score is then deemed optimal. Substitution scores involve assigning a numerical reward for a match and a penalty for a mismatch. An exemplary set of respective match and mismatch scores includes 1, -1; 1, -2; 1, -3; 1, -4; 2, -3; 4, -5, although others are possible. Gap costs involve assigning a numerical penalty for the existence of a gap (insertion or deletion of a nucleotide) and a penalty for extending the width of a gap once it has been formed. Increasing gap costs results in alignments that introduce fewer gaps. An exemplary set of respective costs for the existence and extension of a gap includes 0, -4; -2, -2; -2, -4; -3, -3; -4, -2; -4, -4; -5, -2; -6, -2, although others are possible. In some embodiments, alignment and comparison of the reference sequence and the transgene sequence is performed using default substitution scores and gap penalties, as well as any other default settings provided with the computer software or algorithm for performing the analysis.

[0081] Array Optimization In some embodiments, one or more sequences within an AAV vector can be optimized to improve its functional characteristics compared to the starting reference sequence. For example, without limitation, any protein-coding sequence in a vector can be codon-optimized compared to the wild-type sequence based on the degeneracy of the genetic code and codon usage bias known to exist between different species and between proteins expressed at high or low levels within the same species. Such codon bias can be identified, for example, using a codon adaptation index (CAI) for a particular species. The codon adaptation index (CAI) is described in more detail in Sharp et al., Nucleic Acids Res. 15(3):1281-95 (1987). In some embodiments, the coding sequence is human codon-optimized, meaning that the coding sequence is optimized based on human codon bias. Codon optimization can be facilitated using various algorithms known in the art. As known in the art, different CAIs can be constructed based on the analysis of highly expressed genes, such as human genes. An exemplary human CAI is reported in Haas et al., Curr. Biol. 6(3):315-24 (1996). If desired, protein coding sequences can be codon-optimized for species other than human.

[0082] To increase protein expression levels, different codon optimization strategies have been proposed and implemented. For example, the most frequently used synonymous codons (i.e., those that code for the same amino acid) can be substituted at each position where they do not occur. Alternatively, codon usage can be adjusted throughout the coding sequence to be proportional to the natural codon bias distribution of the host organism. In some embodiments, codon substitution is limited to codons that occur relatively rarely, for example, at a frequency of 10% or less, in highly expressed proteins in a species, as reflected by CAI.

[0083] In some embodiments, protein-coding sequences expressed by AAV vectors of the present disclosure can be codon-optimized by replacing at least one rare codon with a more common synonymous codon. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, and in some embodiments, 100%, of the rare codons in the protein-coding sequence are replaced with a more frequently used synonymous codon, as reflected in a CAI, such as human CAI. In some embodiments, the rare codon occurs at a frequency of 10%, 9%, 8%, 7%, 6%, or 5% or less, as reflected in a CAI, such as human CAI.

[0084] In some embodiments, a protein-coding sequence expressed by an AAV vector of the present disclosure can be codon-optimized by replacing one or more codons with more frequently used synonymous codons as reflected in a CAI, such as the human CAI, such that the calculated CAI value for the overall coding sequence is increased compared to the starting non-codon-optimized sequence, which in some embodiments is the wild-type coding sequence for the protein. Thus, in some embodiments, the CAI value of the starting reference sequence is calculated by reference to a particular CAI lookup table, and one or more codons are replaced with more frequently used synonymous codons such that the overall CAI value of the now codon-optimized coding sequence is at least or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 10.20, 10.21, 10.22, 10.23, 10.24, 10.25, 10.26, 10.27, 10.28, 10.29, 10.30, 10.31, 10.32, 10.33, 10.34, 10.35, 10.36, 10.37, 10.38, 10.39, 10.40, 10.41, 10.42, 10.43, 10.44, 10.45, 10.46, 10.47, 10.48, 10.49, 10.50, 10.51, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.55, 0.60 or 0.70 increase.

[0085] As is known in the art, the presence of hypomethylated CpG dinucleotides in nucleic acids can stimulate an immune response that eliminates transduced cells. Therefore, depleting CpG dinucleotides in vectors can increase the likelihood that vector transduction will result in long-term gene expression (Wright, Mol. Ther. 28(3):701-03(2020)). Taking into account the potentially harmful effects of CpG dinucleotides, in some embodiments, any sequence in a genome, including, for example, enhancers, promoters, introns, open reading frames encoding proteins or functional RNAs, transcription terminators, 5' untranslated regions (UTRs) and / or 3' untranslated region sequences, ITRs, or any other sequences, can be modified to remove one or more CpG dinucleotides, as long as the modified sequence does not unacceptably interfere with or destroy some desired function of the modified element. Because the function of certain elements within a vector, such as ITRs, promoters, and enhancers, can be highly dependent on the identity of specific nucleotides at specific positions, opportunities for significantly depleting such elements of CpG dinucleotides may be more limited. Because AAV vectors of both polarities (e.g., sense and antisense with respect to the coding sequence of a transgene within the genome) are packaged into capsids in approximately equal proportions, CpG depletion strategies may, in some embodiments, involve reducing or eliminating CpG motifs from the nucleotide sequences of vectors of both polarities, as well as vectors containing protein-coding sequences in the sense orientation.

[0086] In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides in the coding sequence or the entire vector sequence (with respect to the sense and / or antisense strand) are deleted or substituted compared to a reference starting sequence, while in other embodiments, at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more CpG dinucleotides, or a range between any of the above values, are deleted or substituted compared to the starting reference sequence. In other embodiments, between 1 and 5, 5 and 10, 10 and 15, 15 and 20, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, 45 and 50, 50 and 55, 55 and 60, 60 and 65, 65 and 70, 70 and 75, 75 and 80, 80 and 85, 85 and 90, 90 and 95, or 95 and 100 CpG dinucleotides are deleted or substituted relative to the reference starting sequence.

[0087] In other embodiments, sequence optimization can increase or decrease the overall GC content compared to a starting reference sequence. Thus, in some embodiments, the overall percentage of G or C nucleotides in a transgene or the entire genome can be increased by at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 percent or more compared to a starting reference sequence, such as a wild-type protein-coding sequence. In other embodiments, the overall percentage of G or C nucleotides in the transgene or the entire genome may be reduced by at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 percent or more as compared to a starting reference sequence, such as a wild-type protein-coding sequence.

[0088] As will be appreciated by those skilled in the art, when optimizing coding sequences, the goal of substituting a more common codon for any particular amino acid in a species (such as human) may be incompatible with other optimization strategies because the introduction of a more frequently used codon may introduce CpG motifs, or the elimination of CpGs may require the use of rarely occurring codons, or codon optimization may increase or decrease GC content in an undesirable manner. In these cases, to achieve improved protein expression, it may be necessary to design and test different optimized coding sequences (encoding the same polypeptide) to identify an acceptable balance between the different optimization strategies.

[0089] In addition to codon bias and CpG content, transgene and vector sequences can be optimized by varying various features. For example, any of the following features that may be found in the sequence and negatively affect transgene expression can be identified (conceptually, such as by using an algorithm, or experimentally) and modified to reduce their effects or eliminated: potential splice sites; premature transcription termination signal sequences (e.g., polyA sequences); translation initiation sites other than the intended initiating methionine (e.g., IRES); GC-rich sequence regions; mRNA 5'-terminal sequences that can form hairpins; and AU-rich elements (AREs) in the mRNA 3'-untranslated region to which destabilizing RNA-binding proteins may bind. Other sequence features that may appear in transgenes and vectors that, when modified, can enhance transgene expression are well known to those skilled in the art.

[0090] In other embodiments, the transgene or vector sequence can be modified to enhance functionality. For example, the original intended start codon in a protein-coding sequence may only be slightly conducive to translation initiation from that site, in which case the surrounding sequence can be altered to conform to the so-called Kozak consensus sequence for translation initiation in eukaryotes (Kozak, Gene 234(2):187-208 (1999)).

[0091] In some embodiments, other types of sequence optimization of a transgene coding sequence, such as (partial or complete) CpG depletion or codon optimization, can improve protein expression from the transgene compared to the same vector containing a non-optimized reference starting sequence, such as the wild-type coding sequence from which the optimized sequence was derived. Thus, for example, an optimized coding sequence of a transgene may be expressed at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more efficiently compared to a non-optimized reference starting sequence, such as the wild-type coding sequence.

[0092] In some embodiments of the nucleic acid molecule of the present disclosure, the nucleic acid molecule comprises a codon-optimized nucleotide sequence encoding a BAG3 polypeptide. In certain embodiments, the codon-optimized nucleotide sequence encoding a BAG3 polypeptide is the nucleotide sequence set forth in SEQ ID NO: 19 (CpG5), SEQ ID NO: 20 (CpG6), SEQ ID NO: 21 (CpG21), SEQ ID NO: 22 (CpG22), SEQ ID NO: 23 (CpG23), or SEQ ID NO: 24 (CpG24), or a functional subsequence, modification, or variant thereof.

[0093] Transcriptional control regions - promoters and enhancers AAV vectors of the present disclosure intended to express a BAG3 protein in and / or from transduced cells may further comprise, as part of the vector's expression cassette, one or more transcriptional control regions operably linked to a transgene encoding a BAG3 polypeptide sequence. As discussed below, different types of transcriptional control regions are known in the art that can be used to control the initiation of transcription of a transgene into RNA. As used herein, the term "operably linked," as well as variants such as "operably linked," "operably linked," and "operably linked," refer to a functional relationship between a transcriptional control region and the transgene such that the transcriptional control region can affect (whether positively or negatively) the transcription of the transgene, without specifying any particular spatial or structural relationship between them. Thus, for example, a transcriptional control region can be operably linked to a transgene whether it is located 5' or 3' to the transgene and / or directly adjacent to the transgene or distal to the transgene. Transcriptional regulatory regions may be constitutively active, active in particular cells or tissues, inducibly active in response to some environmental stimulus, derived from a naturally occurring gene (of any suitable species), modified to improve or alter its function, or even entirely synthetic.

[0094] In some embodiments, the transcriptional control region comprises a promoter region containing the minimal DNA sequence required to initiate transcription by the transcriptional machinery in the transduced cell (e.g., a TATA box or initiation sequence), and often one or more additional proximal elements that act alone or cooperatively to increase the rate of transcription from the basal promoter. Depending on the sequence, a promoter can initiate transcription by RNA polymerase I, RNA polymerase II, or RNA polymerase III; however, AAV vectors intended to express polypeptides such as BAG3 or its variants in the transduced cell often use promoters derived from protein-coding genes that are normally transcribed by RNA pol II.

[0095] In other embodiments, the transcriptional control region includes or further includes at least one enhancer region, which functions to increase the rate of gene transcription beyond that which can be sustained by the basal promoter alone. Enhancers, in their natural context, are often located distally from the promoter of the gene they act on, sometimes tens to hundreds or thousands of base pairs upstream (i.e., 5'), but can also be located elsewhere, such as within an intron or downstream (i.e., 3') of the gene they act on. Promoter regions may include proximal enhancer elements (subsequences that, when removed, can reduce transcription from the basal promoter), but enhancers typically do not include sequences that can function as basal promoters. In nature, enhancer regions are often located distal to the promoters of the genes they act on, but enhancer regions, or enhancer elements from within larger enhancer regions (such elements often correspond to DNA binding sites for transcription factors), when removed from their natural context and repositioned much closer to the promoter, whether from the same gene or even a different gene, may be able to retain at least some of their transcription-promoting function.

[0096] The enhancer and promoter regions of genes described in the scientific literature, when combined with transgenes and other genomic elements required for vector function, can be too large to be accommodated by the packaging capacity of an AAV capsid. Therefore, in some embodiments, methods well known to those skilled in the art can be used to identify functional subsequences within longer enhancer or promoter regions, and then incorporate shorter functional subsequences into transcriptional control regions for use in the vectors of the present disclosure. In this way, the size of transcriptional control regions can be reduced while maintaining their desired function. Using this approach, functional elements from naturally occurring enhancers or promoters can be combined in novel ways, for example, by modifying their number, spacing, and / or positioning, to create hybrid or synthetic enhancers and / or promoters with improved properties. In some embodiments, the enhancer and promoter can each be derived from the same naturally occurring gene, while in other embodiments, the enhancer and promoter can be derived from completely different genes, including genes from different species.

[0097] In some embodiments, in terms of a coding strand (i.e., positive-sense) single-stranded DNA AAV vector, the promoter sequence is located 5' to a downstream sequence to be transcribed into RNA, such as a transgene encoding a protein such as BAG3 or a variant thereof. In some embodiments, an enhancer element or region, if present, may be located 5' to the promoter sequence, or alternatively, may be located elsewhere in the genome, such as in the 5' untranslated region (UTR) or 3' untranslated region adjacent to the transgene, within an intron, 3' to a transcription termination signal sequence, or elsewhere. In some embodiments, a vector may contain two or more enhancer regions (of the same or different types), which may be located adjacent to each other or spaced apart and / or separated by other functional elements in the genome. In some embodiments, the same enhancer element or region is provided as a tandem array of two, three, four, or more repeat units.

[0098] In some embodiments, transcriptional regulatory regions for use in the AAV vectors of the present disclosure are non-tissue-specific, meaning that they are constitutively active in many, but not necessarily all, different cell types. According to some embodiments, non-tissue-specific transcriptional regulatory regions include promoters from certain viruses, such as the human cytomegalovirus major immediate-early gene (CMV-IE), which may contain enhancer elements proximal to the basal promoter (Boshart et al., Cell 41(2):521-30 (1985); Yew et al., Hum. Gene Ther. 8(5):575-84 (1997)), simian virus 40 (SV40), and retroviral long terminal repeat (LTR) promoters from Rous sarcoma virus (RSV) and Moloney murine leukemia virus (MoMLV). In other embodiments, the non-tissue-specific transcriptional control region comprises a promoter (which may include a proximal enhancer element) derived from a different type of animal, such as the human polypeptide chain elongation factor (EF1α) gene, the phosphoglycerate kinase (PGK) gene, the ubiquitin C (UbiC) gene, the chicken beta-actin (CBA) gene, the U1a1 small nuclear RNA promoter or the U1b2 small nuclear RNA promoter (Bartlett et al., Proc. Natl. Acad. Sci. USA 93(17):8852-57 (1996); Wu et al., Mol. Ther. 16(2):280-89 (2008)), or the histone H2 promoter or the histone H3 promoter (Hurt et al., Mol. Cell The genes may be derived from genes that are active in many different cell types (sometimes called "housekeeping" genes), including genes such as ribosomal proteins (e.g., ribosomal proteins), ribosomal proteins (e.g., ribosomal proteins), and ribosomal proteins (e.g., ribosomal proteins).

[0099] Similarly, enhancer regions can be derived from viruses and genes that are active in different cell types from different types of animals. As noted, in some embodiments, promoters and enhancers from the same gene can be combined to create transcriptional control regions for use in the vectors of the present disclosure, although enhancers and promoters from different genes can be combined to create hybrid transcriptional control regions. Commonly used examples include the 1.6 kilobase hybrid enh / pro region, called CAG (or CAGGS), which contains the CMV immediate-early enhancer, the chicken beta actin (CBA) gene promoter, and the CBA intron / exon 1 (Niwa et al., Gene 108(2):193-99(1991)), Ikawa et al., Dev. Growth Differ. 37(4):455-59(1995)), as well as subsequent modifications to reduce its size, in which the CBA intron is replaced with a smaller simian virus 40 (SV40) intron (Wang et al., Gene Ther. 10(26):2105-11(2003)), and the CBA hybrid intron, in which the SV40 intron is replaced with a hybrid intron composed of a 5' donor splice site from the CBA 5'UTR and a 3' acceptor splice site from the MVM intron (CBA This includes what is called CBh (Cellulose-Blocking Hybrid) (Gray et al., Hum. Gene Ther. 22(9):1143-53(2011)).

[0100] In some embodiments, transcriptional control regions for use in the AAV vectors of the present disclosure may be cardiac tissue-specific, meaning that they are more active or most active in directing transgene expression in cell types within the heart compared to cells of other tissues or organs, such as muscle, brain, or liver. In some embodiments, cardiac cell types in which the transcriptional control regions of the AAV vectors of the present disclosure are active include, but are not limited to, cardiomyocytes, although other cell types are possible. Without wishing to be bound by any particular theory of operation, one mechanism by which cardiac tissue or cardiac cell gene transcription specificity may arise is the presence of one or more specific binding sites within the enhancer and / or promoter for a DNA-binding transcriptional activator protein that is preferentially expressed in cardiac cells, such as cardiomyocytes or other cell types within the heart. The use of cardiac tissue or cardiac cell-specific transcriptional control regions may, in some embodiments, be advantageous by reducing or even inhibiting transgene expression in non-cardiac or non-cardiac cells (or other cardiac cell types) that may be transduced by the vector, which may desirably reduce the risk of off-target effects.

[0101] In some embodiments, transcriptional control regions for use in the AAV vectors of the present disclosure are tissue-specific, meaning that they are constitutively active in particular cell types, such as cardiac cell types. Non-limiting examples of cardiac promoters include chicken troponin T (cTNT) (e.g., as in SEQ ID NO: 5), CAG (e.g., as in SEQ ID NO: 28), MHCK7 (e.g., as in SEQ ID NO: 25), CK7 (e.g., as in SEQ ID NO: 27), the endogenous BAG promoter (e.g., as in SEQ ID NO: 26), desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), and cardiac troponin C (TNNC1 or cTnC) promoters, as well as the 600 base pair cardiac troponin T (TNNT2) promoter, or functional subsequences, modifications, or variants thereof.

[0102] Transcription termination sequence In some embodiments, for example, when the transgene comprises a protein-coding sequence (as opposed to a sequence of RNA that has some function other than encoding a protein), the transcription termination sequence may be a polyadenylation signal sequence (variously abbreviated as "polyA," "pA," "poly(A)," or "p(A)"). In some embodiments, polyA signal sequences are derived from naturally occurring genes and can be used in vectors, while in other embodiments, polyA signals can be modified, for example, by shortening them compared to their natural counterparts or by altering their sequence to increase their efficiency in terminating transcription. In other embodiments, the polyA signal may be a hybrid sequence combining polyA sequences from different genes, or a synthetic sequence.

[0103] Non-limiting examples of polyA signals that can be used in the vectors of the present disclosure include polyA signals derived from the bovine growth hormone gene (bGH pA), human, mouse, or rabbit beta-globin genes, SV40 late genes, sNRP1, spA, herpes simplex virus thymidine kinase (HSV TK), or adenovirus type 5 L3 polyadenylation site, among others. In other embodiments, transcription terminators for use in the vectors of the present disclosure include those that terminate RNA transcripts without directing polyadenylation, such as the histone H4 gene mRNA 3' end processing signal (Whitelaw et al., Nucleic Acids Res. 14(17):7059-70 (1986)).

[0104] In some embodiments, the AAV vectors of the present disclosure comprise an expression cassette containing a transgene, the transcription of which is terminated by the inclusion of: (1) a poly(A) site derived from the bovine growth hormone gene (bGH), which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO: 32, or a functional subsequence, modification, or variant thereof; (2) a poly(A) site derived from the SV40 virus, which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof; or (3) a poly(A) site derived from the rabbit beta globin gene, or a functional subsequence, modification, or variant thereof.

[0105] Other vector genome elements In addition to transcriptional control regions and transcription termination signals, other sequences, including cis-regulatory elements, can be included in the genome of the AAV vectors of the present disclosure to improve, control, or regulate transgene expression and / or translation in transduced cells or to confer other functions to the vector. Such elements include, but are not limited to, untranslated regions from the 5' and / or 3' ends of the gene, non-coding exons, introns, splice donor and acceptor sites, lox sites, internal ribosome entry sites (IRES), sequences encoding 2A peptides, elements that stabilize RNA transcripts, binding sites for regulatory miRNAs, microRNA (miRNA) sequences, elements that enhance mRNA nuclear export, including viral post-transcriptional regulatory elements such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and any other elements experimentally shown to improve transgene expression, even though the mechanism may be uncertain. In other embodiments, the vector may contain so-called stuffer or filler sequences, which are intended only to increase the overall length of the vector to a desired size, e.g., to achieve a length that approaches, but is still lower than, the packaging capacity of a particular capsid, thereby reducing the likelihood of accidental packaging of truncated vector or non-vector DNA within the capsid.

[0106] Introns In some embodiments, vectors can include introns to increase transgene expression and / or transcript stability. In some embodiments, protein-coding transgenes are provided in which the exon and intron sequences are identical to those of naturally occurring genes. However, in genes with multiple exons and introns, one or more introns can be removed to minimize overall length while still facilitating the inclusion of other elements, while still maintaining the packaging capacity of the capsid. In other embodiments, introns can be provided from an entirely different gene than the one providing the coding sequence for the vector transgene. Whether the intron is from the same gene as the transgene or a different gene, the intron can be modified from its original sequence, for example, by changing specific nucleotides or by removing internal sequences to shorten its overall length while maintaining splice donor and acceptor sequence motifs required for efficient splicing to occur or other intron cis elements important for function (e.g., enhancers that may be present in the original, unmodified intron sequence). Introns may also be hybrid, in which a splice donor portion of an intron from one gene is paired with a splice acceptor portion of an intron from a different gene, or synthetic, with a sequence that does not correspond to an intron of any known gene. In some embodiments, the intron is located within the coding sequence of the transgene and thus may interrupt it (and may be provided with the donor and acceptor sites necessary for efficient splicing to occur), while in other embodiments, the intron is present but does not interrupt the protein-coding sequence; instead, it is located either 5' or 3' to the coding sequence. If the intron does not interrupt the coding sequence, it may be provided with some exon sequence carried over from its original genetic context, as long as the exon sequence does not contain a potential translation initiation signal. In some embodiments, the intron may be located 3' to the promoter (from the perspective of the positive-strand ssDNA vector) and 5' to the coding sequence.In other embodiments, the intron may be located distally in the vector, either upstream or downstream from the coding sequence.

[0107] Non-limiting examples of introns that can be used in the AAV vectors of the present disclosure, e.g., in the expression cassettes, include introns having the nucleotide sequence of SEQ ID NOs: 6 or 29-31, or functional subsequences, modifications, or variants thereof. Other non-limiting examples include the small intron from minute virus of mice (MVM) (Haut et al., J. Virol. 72(3):183443 (1998), Haut et al., Virology 258(1):8494 (1999)), an internal deletion of intron 1 from human coagulation factor IX (FIXm1 and FIXm2) (Kurachi et al., J. Biol. Chem. 270(10):527681 (1995)), chimeric beta globin splice donor and immunoglobulin heavy chain splice acceptor introns (GenBank U47120.2 nucleotides 890-1022), intron 1 from the mouse alpha globin gene, and the SV40 small t antigen intron, which may comprise or consist of base pairs 4644 to 4552 of GenBank entry J02400.1, and may be modified at positions 4582(g-c), 4580(g-c), 4578(a-c), and 4561(a-t) (Nathwani et al., Blood 107(7):265361 (2006)).

[0108] AAV inverted terminal repeats (ITRs) The ends of the adeno-associated virus genome contain unique nucleotide sequences called inverted terminal repeats (abbreviated as "ITRs") that function as origins of viral DNA replication in infected cells and as priming sites that aid in the conversion of the single-stranded (ssDNA) genome into a double-stranded form (dsDNA) suitable for supporting the transcription of the rep and cap protein-encoding genes. The ITRs also function in packaging the replicated ssDNA genome into AAV capsids. AAV ITRs contain multi-palindromic sequences that can fold back on themselves by intrastrand complementary base pairing to form dsDNA T-shaped hairpin secondary structures.

[0109] As described further below, the expression cassette of an AAV vector of the present disclosure can contain one or more AAV ITRs that function similarly to how they function in the unmodified virus. Unless otherwise specified, the use of the term "inverted terminal repeat" or "ITR" herein includes intact, full-length ITRs and ITRs that have been modified (e.g., truncations, internal deletions, modified sequences (such as trs or D sequences), additions, and substitutions of one or more nucleotides) to retain one or more of the functions attributed to an ITR, including vector rescue from recombinant DNA (such as a plasmid), vector replication, and / or packaging of a vector into assembled capsids, even if less efficiently than an intact ITR of the same type.

[0110] As they exist in packaged viruses and vectors, ITRs located at the 3' end of the ssDNA genome have a free 3' hydroxyl group, while ITRs located at the opposite 5' end of the ssDNA genome have a free 5' end. The 5' ITR is sometimes referred to as the "left" ITR, and the 3' ITR is sometimes referred to as the "right" ITR. However, in plasmids, such as those that can be used for vector production, the vector sequence exists in double-stranded form, resulting in two sets of 5' and 3' ITRs. Therefore, to avoid ambiguity, the strand on which the ITRs are located should be specified to distinguish them. Unless so specified, reference to ITRs in a double-stranded form of a vector, such as a plasmid, refers to the plus or sense strand, i.e., the DNA strand on which the transgene sequence is identical to the coding sequence for the transgene's polypeptide product or, if the transgene does not encode a protein, to the coding sequence for a functional RNA.

[0111] In some non-limiting, merely exemplary embodiments, the expression cassette of an rAAV vector of the present disclosure may comprise one or more ITRs that comprise, consist essentially of, or consist of the nucleotide sequence of any one or more of SEQ ID NO:8 and SEQ ID NO:9, or a functional subsequence, modification, or variant thereof, respectively; other ITR sequences are also possible, or may comprise the complement or reverse complement of any of the sequences specifically listed above.

[0112] In some embodiments, the rAAV vectors disclosed herein comprise at least one 5' ITR, hi some embodiments, at least one 5' ITR comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

[0113] In some embodiments, the rAAV vectors disclosed herein comprise at least one 3' ITR, hi some embodiments, at least one 3' ITR comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

[0114] Stuffer or filler arrangement In some embodiments, the rAAV vectors of the present disclosure further comprise at least one stuffer or filler nucleotide sequence. In some embodiments, the at least one stuffer or filler sequence is an inert nucleotide sequence located within the payload (also known as the expression cassette) from the 5'-ITR to the 3'-ITR. In some embodiments, the at least one stuffer or filler sequence is located 3' of the poly(A) signal sequence and 5' of the ITR. The at least one stuffer or filler sequence can have any desired design, and the use of a stuffer or filler sequence as defined herein is intended to result in a reduction of contaminating nucleic acids packaged into viral particles / capsids. In some embodiments, the stuffer or filler sequence can be a random sequence of nucleotides. In some embodiments, the at least one stuffer or filler sequence is derived from a human sequence. In some embodiments, the stuffer or filler sequence is derived from an intron of a gene, such as the Factor 9 gene or the gene encoding the human or other species TATA box binding protein (TBP), or a functional subsequence, modification, or variant thereof. In some embodiments, the stuffer or filler sequence is a TBP gene intron, or a functional subsequence, modification, or variant thereof. In some embodiments, at least one stuffer or filler sequence can be one or more intron sequences modified, for example, to deplete CpGs. In some embodiments, the stuffer or filler sequence has 97% sequence identity to intron 1 of the Factor 9 gene. In some embodiments, the stuffer or filler sequence has the nucleotide sequence of SEQ ID NO: 11.

[0115] Spacer In some embodiments, the rAAV vector plasmid of the present disclosure comprises at least one spacer sequence. A spacer is an inert nucleotide sequence located outside the 5'-ITR to 3'-ITR payload (also known as the expression cassette); i.e., the spacer is part of the rAAV vector plasmid. The spacer can have any desired design, and use of the spacer sequence defined herein is intended to result in reduced contaminating nucleic acid packaging into viral particles / capsids. In some embodiments, the spacer sequence can be a random sequence of nucleotides. In other embodiments, it can encode a gene product, such as a marker gene. In certain embodiments, the spacer can be, for example, one or more intron sequences from the Factor 9 gene modified to deplete CpGs. In some embodiments, the length of the spacer can range from about 10 nucleotides to about 10,000 nucleotides, e.g., from about 100 nucleotides to about 8,000 nucleotides. In certain embodiments, the spacer sequence is less than 2,000 nucleotides in length.

[0116] In some embodiments, the rAAV vector plasmids disclosed herein comprise at least one left (i.e., 5' of the 5' ITR) spacer sequence. In some embodiments, the at least one left spacer comprises the nucleotide sequence of SEQ ID NO: 12, or a functional subsequence, modification, or variant thereof.

[0117] In some embodiments, the rAAV vector plasmids disclosed herein comprise at least one right (i.e., 3' of the 3' ITR) spacer sequence. In some embodiments, the at least one right spacer comprises the nucleotide sequence of SEQ ID NO: 13, or a functional subsequence, modification, or variant thereof.

[0118] In some embodiments, the rAAV vector plasmids of the present disclosure containing the expression cassettes should be greater than about 4.7 kilobases, e.g., greater than about 5 kilobases, hi some embodiments, the rAAV vector plasmids of the present disclosure containing the expression cassettes range in size from about 4.7 kilobases to about 12 kilobases.

[0119] In some embodiments, the rAAV vector plasmid comprises at least one left spacer and / or at least one right spacer, such that the at least one left spacer and / or at least one right spacer increases the overall length of the rAAV vector plasmid to about 4.7 to about 12 kilobases, e.g., about 9 to about 10 kilobases.

[0120] Compound A Some embodiments include Compound A, an AAV9 gene therapy vector that delivers BAG3 protein expression to the heart under the control of a cardiac-specific promoter (e.g., the troponin T (cTNT) promoter). It is intended for the treatment of diseases or disorders associated with 1) mutations in Bcl2-associated athogen 3 (BAG3) or (2) mediated by or associated with BAG3 protein expression. Compound A is a cardiac-directed gene therapy, and AAV9 is known to transduce the heart in mice, non-human primates, and humans. In addition, the cTNT promoter confers strongly cardiac-biased transgene expression (Prasad et al., Gene Ther. 18(1):43-52 (2011)). In some embodiments, a single intravenous administration can be used to transduce a significant proportion of cardiac cardiomyocytes. BAG3 cKO mice exhibit substantial defects in left ventricular ejection fraction and left ventricular end-diastolic volume at 18-21 weeks of age. Intravenous administration of Compound A at 1E13, 3E13, and 9E13 vg / kg to 12-week-old BAG3 cKO mice with low and impaired cardiac function resulted in dose-dependent improvements in cardiac function (ejection fraction) and structure (diastolic volume) compared with untreated cKO controls. Mid- and high-dose levels stabilized ejection fraction over time. Analysis of BAG3 mRNA biodistribution in the heart by in situ hybridization showed that the effect on cardiac function increased with increasing cardiac coverage, reaching a plateau at 20-80% coverage. Analysis of BAG3 protein levels in the heart similarly showed an increase in efficacy from 0% to 20% of wild-type BAG3 levels and a plateau at 20-120% coverage. These correlations defined the cardiac biodistribution and expression targets required for cardiac efficacy.

[0121] In biodistribution and safety studies in non-human primates, human BAG3 protein levels in the heart exceeded the target threshold of 20% of wild-type (WT) at 1.3E14 vg / kg, and the percentage of human BAG3 mRNA-positive cardiomyocytes in the heart exceeded the target threshold of about 20% at about 4E13 and about 1.3E14 vg / kg. In some embodiments, clinically effective doses are predicted to be about 3E13 to about 1E14 vg / kg, inclusive. No safety-limiting findings were observed.

[0122] Compound A is intended as an AAV gene therapy for heart-related diseases and disorders, such as BAG3 DCM. This vector is distinguished from other vectors through its intravenous delivery (broader and more uniform cardiac transduction than intracoronary arteries) and use of a cardiac-specific promoter (e.g., greater safety and potentially more durable expression than CMV).

[0123] Pharmaceutical Compositions In some embodiments, the present disclosure provides pharmaceutical compositions or medicaments for preventing or treating a disease, disorder, or condition mediated by or associated with decreased expression and / or activity of BAG3, e.g., dilated cardiomyopathy. In some embodiments, the pharmaceutical composition comprises a modified nucleic acid, a recombinant nucleic acid, a viral vector genome, an expression vector, a host cell, or an rAAV vector, and a pharmaceutically acceptable carrier.

[0124] In some embodiments, the pharmaceutical composition comprises a vector (e.g., a viral vector genome, an expression vector, an rAAV vector) or a host cell comprising a therapeutically effective amount of a modified nucleic acid encoding BAG3 that can increase the expression and / or activity level of BAG3 in the cell.

[0125] The rAAV vector or vector genome dose to achieve a therapeutic effect, e.g., the dose in vector genomes / kilogram of body weight (vg / kg), will vary based on several factors, including, but not limited to, the route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the particular disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. Generally, the dose will be in the range of at least 1E8, or more, e.g., 1E9, 1E10, 1E11, 1E12, 1E13, or 1E14, 1E15, 1E16, 1E17, or more, vector genomes per kilogram of subject body weight (vg / kg) to achieve a therapeutic effect. In some embodiments, a pharmaceutically effective amount of rAAV vector is in the range of about 1E10 to about 1E17 vector genomes per kilogram of subject body weight (vg / kg).

[0126] In some embodiments, the pharmaceutical composition comprises a vector (e.g., a viral vector genome, an expression vector, an rAAV vector) or a host cell (e.g., for ex vivo gene therapy) comprising a therapeutically effective amount of a nucleic acid encoding BAG3, and the composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, other medicinal agent, or a combination thereof. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other medicinal agent is not biologically or otherwise undesirable, e.g., a substance may be administered to a subject without causing undesirable biological effects that outweigh the beneficial biological effects of the substance.

[0127] Any suitable pharmaceutically acceptable carrier or excipient can be used in preparing a pharmaceutical composition according to the present invention (see, for example, Remington The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997).

[0128] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluids), microemulsions, liposomes, or other ordered structures suitable for accommodating high product (e.g., viral vector particle, microparticle, or nanoparticle) concentrations. In some embodiments, pharmaceutical compositions comprising the modified nucleic acids of the present disclosure, vector genomes comprising the modified nucleic acids, host cells, or rAAV vectors are formulated in water or buffered saline. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. In some embodiments, the nucleic acids, vectors, and / or host cells of the disclosure can be administered in a controlled release formulation, for example, in compositions which include slow release polymers or other carriers which will protect the product against rapid release, including implants and microencapsulated delivery systems.

[0129] In some embodiments, pharmaceutical compositions of the present disclosure are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular (e.g., cardiac), and / or intraarticular administration. In some embodiments, pharmaceutical compositions comprising an rAAV vector comprising a nucleic acid encoding BAG3 are formulated for administration by intravenous injection.

[0130] In some embodiments, the pharmaceutical compositions of the present disclosure comprise at least one pharmaceutically acceptable salt in an amount ranging from about 1 mM to about 450 mM, or from about 2 mM to about 350 mM, or from about 20 mM to about 200 mM. In certain embodiments, the at least one pharmaceutically acceptable salt is present in the pharmaceutical composition at about 150 mM. In other embodiments, the at least one pharmaceutically acceptable salt is present in the pharmaceutical composition at about 350 mM. In certain embodiments, the at least one pharmaceutically acceptable salt is sodium chloride, magnesium chloride, potassium chloride, calcium chloride, calcium phosphate, or a combination thereof. In certain embodiments, the at least one pharmaceutically acceptable salt comprises sodium chloride and magnesium chloride. In certain embodiments, the at least one pharmaceutically acceptable salt comprises about 5 mM to about 6 mM sodium chloride and about 45 mM to about 55 mM magnesium chloride. In other embodiments, the at least one pharmaceutically acceptable salt comprises sodium chloride and potassium chloride. In another embodiment, the at least one pharmaceutically acceptable salt comprises about 300 mM to about 400 mM sodium chloride and about 2 mM to about 3 mM potassium chloride.

[0131] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise at least one buffering agent. In certain embodiments, the buffering agent is citrate, histidine, acetate, phosphate, Tris hydrochloride, tromethamine, or a combination thereof. In one embodiment, the buffering agent is phosphate (e.g., about 5 mM to about 15 mM). In another embodiment, the buffering agent is Tris hydrochloride (e.g., about 2 mg / mL to about 3 mg / mL) and tromethamine (about 0.25 mg / mL to about 0.75 mg / mL). In certain embodiments, the at least one buffering agent is present in an amount ranging from about 10 mM to about 40 mM. In some embodiments, the at least one buffering agent is present in an amount of about 20 mM.

[0132] In some embodiments, the pharmaceutical composition of the present disclosure further comprises at least one cryoprotectant. In certain embodiments, the at least one cryoprotectant is a sugar or sugar alcohol. In some embodiments, the at least one cryoprotectant is trehalose, sucrose, sorbitol, mannitol, or a combination thereof. In one embodiment, the at least one cryoprotectant is sucrose (e.g., about 30 mg / mL to about 50 mg / mL). In another embodiment, the at least one cryoprotectant is sorbitol (e.g., about 2% to about 10%). In certain embodiments, the at least one cryoprotectant is present in an amount of up to about 20%. In some embodiments, the at least one cryoprotectant is present in an amount ranging from about 3% to about 15%. In some embodiments, the at least one cryoprotectant is present in an amount of about 4% or about 5%.

[0133] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise at least one surfactant. In certain embodiments, the at least one surfactant is a polaxamer or a polysorbate. In some embodiments, the at least one surfactant is polaxamer 188, polysorbate 20, or polysorbate 80. In some embodiments, the at least one surfactant is about 0.1% to about 0.5% polaxamer 188. In certain embodiments, the at least one surfactant is present in an amount ranging from about 0.0001% to about 1%. In one embodiment, the at least one surfactant is present in an amount of about 0.02%. In another embodiment, the at least one surfactant is present in an amount of about 0.002%.

[0134] In some embodiments, pharmaceutical compositions of the present disclosure have a pH ranging from about 6 to about 8. In some embodiments, pharmaceutical compositions have a pH ranging from about 7 to about 8. In one embodiment, a pharmaceutical composition has a pH of about 7.6. In another embodiment, a pharmaceutical composition has a pH of about 7.4.

[0135] In some embodiments, a rAAV vector containing a BAG3 transgene is purified and suspended in a formulation containing 10 mM phosphate, 350 mM NaCl, 2.7 mM KCl, 5% sorbitol, and 0.002% Pluronic at pH 7.4. In some embodiments, a rAAV vector containing a BAG3 transgene is purified and suspended in a formulation containing 0.47 mg / mL tromethamine, 2.54 mg / mL Tris (Tris and tromethamine together are 20 mM), 10.17 mg / mL MgCl (50 mM), 5.84 mg / mL NaCl (100 mM), 40 mg / mL sucrose (4%), and 0.2 mg / mL polaxamer 188 (0.02%) at pH 7.6.

[0136] AAV vector production As is known in the art, AAV vectors can be produced in a variety of ways, including on a large scale. AAV vectors can be produced in mammalian or insect cells, for example, and then purified. A traditional approach that does not rely on coinfection with a helper virus involves the use of three plasmids, as discussed above. One plasmid contains genes for helper virus factors, a second plasmid contains the AAV genome sequence in double-stranded form, and a third plasmid contains the AAV rep and cap genes. In practice, the three plasmids are often replicated separately in bacteria, purified, mixed together in solution in a predetermined ratio, and then mixed with a transfection agent. The transfection mixture is then used to transfect suitable mammalian host cells (in adherent or suspension cell culture), which are incubated for a sufficient time (e.g., 48-72 hours) and under sufficient conditions for the host cells to express the helper factors and rep and cap genes, and for the AAV vector to replicate from its plasmid template and be packaged into capsids. In some embodiments, the host cells are HEK293 cells or their derivatives, which constitutively express AdV helper factors E1A and E1B; therefore, the helper plasmid need only contain the AdV E2A, E4ORF6, and VA RNA genes. If other mammalian host cells that do not themselves produce AdV or other viral helper factors are used, the use of helper plasmids containing the missing or otherwise required helper factors is necessary. While the so-called triple transfection method described above is commonly used, it is not necessary for the genes for the helper factors and the rep and cap genes to be provided on separate plasmids. In principle, all of these genes can be housed, for example, on one plasmid, in which case two plasmids can be used for transfection.

[0137] In search of a more efficient method for large-scale production of AAV vectors, stable cell lines have been generated that contain some, but not all, of the components that would normally be required for transient transfection. Packaging cell lines contain stably integrated AAV rep and cap genes. AAV production in packaging cells requires transient transfection of the cells with a plasmid containing an AAV vector and infection with a helper virus. Alternatively, AAV vectors can be produced in packaging cells without transfection by first infecting the cells with AdV (either wild-type or E2b gene-deleted AdV) that provides the AdV E1 gene products that drive rep and cap expression in the cells, as well as helper factors required for AAV replication, and then infecting them with a replication-deficient hybrid AdV in which the AAV vector replaces the E1 gene in the hybrid virus genome.

[0138] Alternatively, the producer cell line also contains stably integrated AAV rep and cap genes and the AAV vector. AAV production in the producer cells requires infection of these cells with a helper virus. Packaging and producer cells have been described (Martin et al., Hum. Gene Ther. Methods 24(4):253-69 (2013); Gao et al., Hum. Gene Ther. 9(16):2353-62 (1998); Clement et al., Mol. Ther. Methods Clin. Dev. 3:16002 (2016)). Other cell lines are contemplated for producing AAV vectors in mammalian cells, including on a commercial scale.

[0139] The baculovirus system has also been used to produce AAV vectors, in which Sf9 insect cells are infected with recombinant baculovirus vectors that variously contain the AAV rep and cap genes and the AAV genome. The exogenous gene is expressed, and the genome is then packaged into vector particles within the cell. In early versions of this system, each component, rep, cap, and genome, was carried by three separate baculoviruses. Subsequent modifications included combining rep and cap into a single baculovirus, so that only two types of baculovirus were required, and generating the Sf9 cell line, which contains stably integrated AAV rep and cap genes and requires only infection with a single type of recombinant baculovirus containing the AAV vector (Urabe et al., Hum. Gene Ther. 13(16):1935-43 (2002); Virag et al., Hum. Gene Ther. 20(8):807-17 (2009); Smith et al., Mol. Ther. 17(11):1888-96 (2009); Mietzsch et al., Hum. Gene Ther. 25(3):212-22 (2014)). Other cell lines are contemplated for producing AAV vectors in insect cells, including on a commercial scale.

[0140] host cell As used herein, "host cells" refer to cells suitable or adapted for in vitro production of AAV vectors. Host cells are often clonal cell lines capable of dividing for multiple generations before aging causes growth arrest, or they may even be immortal. To produce vectors, host cells can be transiently or non-transiently modified by the introduction of exogenous genetic information designed to direct the biosynthesis in the host cell of various components required for AAV vector assembly, particularly AAV capsid proteins, Rep proteins, helper virus factors, and vectors. For example, host cells can be transfected with exogenously supplied nucleic acids, such as in the form of one or more DNA plasmids, containing nucleotide sequences encoding the necessary vector components.

[0141] Various methods for transfecting host cells with nucleic acids are known in the art. These methods include, but are not limited to, mixing the nucleic acid with certain compounds that can form complexes with the nucleic acid and then be taken up by the cell, such as calcium phosphate or cationic organic compounds (e.g., DEAE-dextran, polyethylenimine (PEI), polylysine, polyornithine, polybrene, cyclodextrins, cationic lipids, and others known in the art. Transfection can also be performed non-chemically by electroporation and more exotic techniques, such as biolistic particle delivery. As known in the art, transfection can be transient or stable. In transient transfection, the transfected nucleic acid is present in the cell for a limited period of time and, in the case of DNA, does not integrate into the genome. In stable transfection, the DNA introduced into the cell is expressed as an episomal plasmid. Stably transfected cells can persist for long periods of time, either as a single vector or integrated into a chromosome. Stably transfected cells are typically produced by transfecting cells with a plasmid containing a nucleotide sequence encoding a selectable marker gene and one or more necessary vector components, and then growing and maintaining the cells under selection, i.e., conditions under which untransfected cells or transfected cells in which the exogenous DNA, including the selectable marker, has been lost for some reason, are killed. For example, the plasmid may contain an antibiotic resistance gene, and transfected cells can be selected by adding an antibiotic to the medium in which the cells are grown. In some embodiments, the nucleotide sequence introduced into a stably transfected host cell and encoding one or more of the necessary vector components is under the control of an inducible promoter, such that it is not expressed, or is expressed only at low levels, unless an environmental factor, such as a drug, metal ion, or elevated temperature, is introduced that induces the promoter as the cells grow.

[0142] In other embodiments, genetic engineering methods, such as knock-in or gene editing, can be used to modify the host cell genome in a non-transient and targeted manner to direct the host cell to produce one or more of the required vector components. In other embodiments, nucleotide sequences encoding one or more of the required vector components can be introduced into host cells for the purpose of directing the production of AAV vectors by transduction, where the host cells are infected with a modified virus containing such nucleotide sequences. Examples of viral vectors useful for this purpose include adenoviruses, retroviruses (including lentiviruses), baculoviruses, vaccinia viruses, and herpes simplex viruses, among others.

[0143] Host cells can be any type of cell known in the art to be useful for producing AAV vectors. Host cells are often animal cells and can be of various types or species, such as insect cells or mammalian cells, including rat, mouse, or human cells, among others. In some embodiments, host cells useful for producing the AAV vectors of the disclosure are mammalian host cells, such as HeLa cells, Cos cells, HEK293 cells (and variants of HEK293 cells such as HEK293E cells, HEK293F cells, HEK293H cells, HEK293T cells, or HEK293FT cells), A549 cells, BHK cells, Vero cells, NIH 3T3 cells, HT-1080 cells, Sp2 / 0 cells, NS0 cells, C127 cells, AGE1.HN cells, CAP cells, HKB-11 cells, WI-38 cells, MRC-5 cells, or PER.C6 cells, among many others. In some embodiments, host cells useful for producing the AAV vectors of the invention are insect host cells, such as Sf9, ExpiSf9, ​​Sf21, S2, D.Mel2, Tn-368, or BTI-Tn-5B1-4 cells, among many others. In some embodiments, host cells, including but not limited to HEK293 cells and variants thereof, can be adapted for growth in suspension culture.

[0144] For the purpose of producing AAV vectors, host cells are grown or maintained in culture under controlled conditions conducive to host cell growth and vector biosynthesis. For example, host cells can be grown in a chemically defined liquid medium that provides all nutrients necessary for cell growth and biosynthesis. Exemplary media include DMEM, DMEM / F12, MEM, and RPMI 1640 for mammalian host cells, and Express Five SFM, Sf-900 II SFM, Sf-900 III, or ExpiSf CD for certain insect cells. Such media may be supplemented with antibiotics, growth factors, or cytokines (recombinantly produced or present in animal serum, such as FBS) known to stimulate growth of the particular type of cell being used, as well as other components that may be required for optimal biosynthesis of AAV vectors but are naturally in low supply. Exemplary supplements include essential amino acids, glutamine, vitamin K, insulin, BSA, or transferrin. In addition to the growth medium, other culture conditions, such as pH, temperature, and CO2 and oxygen concentrations, may be controlled to optimize cell growth and / or productivity.

[0145] Host cells in culture can be grown or maintained in a number of vessels known in the art, such as stirred tank bioreactors, wave bags, spinner flasks, hollow fiber bioreactors, or roller bottles, some of which can be designed and configured for single or multiple use. Depending on the characteristics of the host cells in question, host cells can be grown in adherent cell culture, in which cells attach to and grow in contact with a physical substrate, or in suspension cell culture, in which single cells either float freely in the medium that sustains them or attach to bead microcarriers suspended in the medium. As known in the art, various techniques have been developed to grow host cells to high cell densities, such as perfusion culture, which can increase the total amount of AAV vector produced in a single production run.

[0146] As is known in the art, samples of host cells are often maintained in frozen cell banks, such as master cell banks and working cell banks, facilitating the production of biological products in multiple batches over time while ensuring consistent performance by the host cells. Prior to AAV vector production activities, frozen samples of host cells from the cell bank are typically thawed, seeded into small culture volumes, and grown to further increase in density or number, with increasing culture volumes. When the host cells reach a desired cell density and / or volume in culture, exogenous genetic material can be introduced, such as by transfection with plasmid DNA or infection or transduction with a viral vector, to cause the host cells to begin producing AAV vectors. Alternatively, when using host cells that have been non-transiently modified and in which a nucleotide sequence encoding one or more required vector components is under inducible control, environmental factors necessary to induce expression can be introduced. The host cells can then be grown or maintained in culture for a time and under conditions sufficient to produce the AAV vector.

[0147] Embodiment Embodiment 1: A nucleic acid molecule comprising a nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof.

[0148] Embodiment 2: The nucleic acid molecule of embodiment 1, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is a codon-optimized nucleotide sequence.

[0149] Embodiment 3: The nucleic acid molecule of embodiment 1 or 2, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0150] Embodiment 4: The nucleic acid molecule of any one of embodiments 1 to 3, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0151] Embodiment 5: The nucleic acid molecule of any one of embodiments 1 to 4, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0152] Embodiment 6: The nucleic acid molecule of any one of embodiments 1 to 5, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0153] Embodiment 7: The nucleic acid molecule of any one of embodiments 1 to 6, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0154] Embodiment 8: The nucleic acid molecule of any one of embodiments 1 to 7, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0155] Embodiment 9: The nucleic acid molecule of any one of embodiments 1 to 8, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0156] Embodiment 10: The nucleic acid molecule according to any one of embodiments 1 to 9, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0157] Embodiment 11: A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid molecule of any one of embodiments 1 to 10.

[0158] Embodiment 12: The rAAV vector of embodiment 11, wherein the rAAV vector comprises a serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

[0159] Embodiment 13: The rAAV vector of embodiment 11 or 12, wherein the rAAV vector comprises an AAV9 capsid protein.

[0160] Embodiment 14: The rAAV vector of embodiment 13, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof; a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof; or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

[0161] Embodiment 15: The rAAV vector of embodiment 13 or 14, wherein the AAV9 capsid proteins include a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof; a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof; and a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

[0162] Embodiment 16: The rAAV vector of any one of embodiments 11 to 15, wherein the rAAV vector further comprises at least one cardiac promoter operably linked to a nucleic acid molecule comprising a nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof.

[0163] Embodiment 17: The rAAV vector of embodiment 16, wherein the at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant thereof, respectively.

[0164] Embodiment 18: The rAAV vector of embodiment 16 or 17, wherein at least one cardiac promoter is a cTNT promoter, or a functional subsequence, modification, or variant thereof.

[0165] Embodiment 19: The rAAV vector of embodiment 17 or 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

[0166] Embodiment 20: The rAAV vector of embodiment 17 or 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO:5.

[0167] Embodiment 21: The rAAV vector of any one of embodiments 11 to 20, wherein the rAAV vector further comprises at least one intron.

[0168] Embodiment 22: The rAAV vector of embodiment 21, wherein the intron comprises the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, or a functional subsequence, modification, or variant thereof.

[0169] Embodiment 23: The rAAV vector of embodiment 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO:6.

[0170] Embodiment 24: The rAAV vector of embodiment 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 29.

[0171] Embodiment 25: The rAAV vector of embodiment 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 30.

[0172] Embodiment 26: The rAAV vector of embodiment 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 31.

[0173] Embodiment 27: The rAAV vector of any one of embodiments 11 to 26, wherein the rAAV vector further comprises at least one 5' inverted terminal repeat (ITR) sequence.

[0174] Embodiment 28: The rAAV vector of embodiment 27, wherein at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

[0175] Embodiment 29: The rAAV vector of embodiment 27 or 28, wherein at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO:8.

[0176] Embodiment 30: The rAAV vector of any one of embodiments 11 to 29, wherein the rAAV vector further comprises at least one 3' ITR sequence.

[0177] Embodiment 31: The rAAV vector of embodiment 30, wherein at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

[0178] Embodiment 32: The rAAV vector of embodiment 30 or 31, wherein at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:9.

[0179] Embodiment 33: The rAAV vector of any one of embodiments 11 to 32, wherein the rAAV vector further comprises at least one transcription termination sequence.

[0180] Embodiment 34: The rAAV vector of embodiment 33, wherein at least one transcription termination sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

[0181] Embodiment 35: The rAAV vector of embodiment 33 or 34, wherein at least one transcription termination sequence is an SV40 polyA sequence.

[0182] Embodiment 36: The rAAV vector of any one of embodiments 33 to 35, wherein at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

[0183] Embodiment 37: The rAAV vector of any one of embodiments 33 to 36, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0184] Embodiment 38: The rAAV vector of embodiment 33 or 34, wherein at least one transcription termination sequence is a BGH polyA sequence.

[0185] Embodiment 39: The rAAV vector of any one of embodiments 33, 34, or 38, wherein at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 32, or a functional subsequence, modification, or variant thereof.

[0186] Embodiment 40: The rAAV vector of any one of embodiments 33, 34, 38, or 39, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 32.

[0187] Embodiment 41: The rAAV vector of any one of embodiments 11 to 40, wherein the rAAV vector further comprises at least one stuffer or filler sequence, preferably at least one stuffer or filler sequence including an ITR sequence, increasing the overall length of the nucleic acid molecule of the rAAV vector to approximately 4.2 to 4.7 kilobases.

[0188] Embodiment 42: The rAAV vector of embodiment 41, wherein at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11, or a functional subsequence, modification, or variant thereof.

[0189] Embodiment 43: The rAAV vector of embodiment 41 or 42, wherein at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11.

[0190] Embodiment 44: An rAAV vector comprising, in 5' to 3' order: (a) at least one 5' ITR sequence; (b) at least one cardiac promoter; (c) at least one intron; (d) at least one nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof, operably linked to at least one cardiac promoter; (e) at least one transcription termination sequence; (f) at least one stuffer or filler sequence; (g) at least one 3' ITR sequence.

[0191] Embodiment 45: The rAAV vector of embodiment 44, wherein the rAAV vector comprises a serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

[0192] Embodiment 46: The rAAV vector of embodiment 44 or 45, wherein the rAAV vector comprises an AAV9 capsid protein.

[0193] Embodiment 47: The rAAV vector of embodiment 46, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof; a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof; or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

[0194] Embodiment 48: The rAAV vector of embodiment 46 or 47, wherein the AAV9 capsid proteins include a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof, a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof, and a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

[0195] Embodiment 49: An rAAV vector plasmid comprising, in 5' to 3' order: (a) at least one left spacer sequence; (b) at least one 5' ITR sequence; (c) at least one cardiac promoter; (d) at least one intron; (e) at least one nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof, operably linked to at least one cardiac promoter; (f) at least one transcription termination sequence; (g) at least one stuffer or filler sequence; (h) at least one 3' ITR sequence; (i) at least one right spacer sequence; and

[0196] Embodiment 50: The rAAV vector of any one of embodiments 44 to 48, or the rAAV vector plasmid of embodiment 49, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is a codon-optimized nucleotide sequence.

[0197] Embodiment 51: The rAAV vector of any one of embodiments 44 to 48 or 50, or the rAAV vector plasmid of any one of embodiments 49 or 50, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0198] Embodiment 52: The rAAV vector of any one of embodiments 44-48, 50, or 51, or the rAAV vector plasmid of any one of embodiments 49-51, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

[0199] Embodiment 53: The rAAV vector of any one of embodiments 44 to 48 or 50 to 52, or the rAAV vector plasmid of any one of embodiments 49 to 52, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0200] Embodiment 54: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 53, or an rAAV vector plasmid described in any one of embodiments 49 to 53, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0201] Embodiment 55: The rAAV vector of any one of embodiments 44 to 48 or 50 to 54, or the rAAV vector plasmid of any one of embodiments 49 to 54, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0202] Embodiment 56: The rAAV vector of any one of embodiments 44 to 48 or 50 to 55, or the rAAV vector plasmid of any one of embodiments 49 to 55, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0203] Embodiment 57: The rAAV vector of any one of embodiments 44 to 48 or 50 to 56, or the rAAV vector plasmid of any one of embodiments 49 to 56, wherein at least one nucleotide sequence encoding a BAG3 polypeptide, or a variant thereof, is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0204] Embodiment 58: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 57, or an rAAV vector plasmid described in any one of embodiments 49 to 56, wherein at least one nucleotide sequence encoding a BAG3 polypeptide or a variant thereof is the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

[0205] Embodiment 59: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 58, or an rAAV vector plasmid described in any one of embodiments 49 to 58, wherein at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

[0206] Embodiment 60: The rAAV vector of any one of embodiments 44 to 48 or 50 to 59, or the rAAV vector plasmid of any one of embodiments 49 to 59, wherein at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8.

[0207] Embodiment 61: The rAAV vector of any one of embodiments 44 to 48 or 50 to 60, or the rAAV vector plasmid of any one of embodiments 49 to 60, wherein at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant thereof.

[0208] Embodiment 62: The rAAV vector or rAAV vector plasmid of embodiment 61, wherein at least one cardiac promoter is a cTNT promoter, or a functional subsequence, modification, or variant thereof.

[0209] Embodiment 63: The rAAV vector or rAAV vector plasmid of embodiment 61 or 62, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

[0210] Embodiment 64: The rAAV vector or rAAV vector plasmid of any one of embodiments 61 to 63, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5.

[0211] Embodiment 65: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 64, or an rAAV vector plasmid described in any one of embodiments 49 to 64, wherein at least one intron comprises the nucleotide sequence of SEQ ID NO: 6, or a functional subsequence, modification, or variant thereof.

[0212] Embodiment 66: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 65, or an rAAV vector plasmid described in any one of embodiments 49 to 65, wherein at least one intron comprises the nucleotide sequence of SEQ ID NO: 6.

[0213] Embodiment 67: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 66, or an rAAV vector plasmid described in any one of embodiments 49 to 66, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 29.

[0214] Embodiment 68: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 67, or an rAAV vector plasmid described in any one of embodiments 49 to 67, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 30.

[0215] Embodiment 69: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 68, or an rAAV vector plasmid described in any one of embodiments 49 to 68, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 31.

[0216] Embodiment 70: The rAAV vector of any one of embodiments 44 to 48 or 50 to 69, or the rAAV vector plasmid of any one of embodiments 49 to 69, wherein at least one transcription termination sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

[0217] Embodiment 71: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 70, or an rAAV vector plasmid described in any one of embodiments 49 to 70, wherein at least one transcription termination sequence is an SV40 polyA sequence.

[0218] Embodiment 72: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 71, or an rAAV vector plasmid described in any one of embodiments 49 to 71, wherein at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

[0219] Embodiment 73: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 72, or an rAAV vector plasmid described in any one of embodiments 49 to 72, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0220] Embodiment 74: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 70, or an rAAV vector plasmid described in any one of embodiments 49 to 70, wherein at least one transcription termination sequence is a BGH polyA sequence.

[0221] Embodiment 75: The rAAV vector of any one of embodiments 44-48, 50-70, or 74, or the rAAV vector plasmid of any one of embodiments 49-70, or 74, wherein at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 32, or a functional subsequence, modification, or variant thereof.

[0222] Embodiment 76: The rAAV vector of any one of embodiments 44 to 48, 50 to 70, 74, or 75, or the rAAV vector plasmid of any one of embodiments 49 to 70, 74, or 75, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 32.

[0223] Embodiment 77: The rAAV vector of any one of embodiments 44 to 48 or 50 to 76, or the rAAV vector plasmid of any one of embodiments 49 to 76, wherein at least one stuffer or filler sequence, including ITR sequences and excluding spacer sequences, increases the overall length of the nucleic acid molecule of the rAAV vector or rAAV vector plasmid to approximately 4.2 to 4.7 kilobases.

[0224] Embodiment 78: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 77, or an rAAV vector plasmid described in any one of embodiments 49 to 77, wherein at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11, or a functional subsequence, modification, or variant thereof.

[0225] Embodiment 79: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 78, or an rAAV vector plasmid described in any one of embodiments 49 to 78, wherein at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11.

[0226] Embodiment 80: The rAAV vector of any one of embodiments 44 to 48 or 50 to 79, or the rAAV vector plasmid of any one of embodiments 49 to 79, wherein at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

[0227] Embodiment 81: An rAAV vector described in any one of embodiments 44 to 48 or 50 to 80, or an rAAV vector plasmid described in any one of embodiments 49 to 80, wherein at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9.

[0228] Embodiment 82: The rAAV vector plasmid of any one of embodiments 49 to 81, wherein at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO: 12, or a functional subsequence, modification, or variant thereof.

[0229] Embodiment 83: The rAAV vector plasmid of any one of embodiments 49 to 82, wherein at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO: 12.

[0230] Embodiment 84: The rAAV vector plasmid of any one of embodiments 49 to 83, wherein at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO: 13, or a functional subsequence, modification, or variant thereof.

[0231] Embodiment 85: The rAAV vector plasmid of any one of embodiments 49 to 84, wherein at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO: 13.

[0232] Embodiment 86: The rAAV vector plasmid of any one of embodiments 49 to 85, wherein the rAAV vector plasmid further comprises at least one left spacer and / or at least one right spacer, preferably wherein the at least one left spacer and / or at least one right spacer increases the overall length of the rAAV vector plasmid to approximately 4.7 to 12 kilobases, preferably approximately 9 to 10 kilobases.

[0233] Embodiment 87: An rAAV vector described in any one of embodiments 11 to 48 or 50 to 81, or an rAAV vector plasmid described in any one of embodiments 49 to 86, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 14, or a functional subsequence, modification, or variant thereof.

[0234] Embodiment 88: An rAAV vector described in any one of embodiments 11 to 48, 50 to 81, or 87, or an rAAV vector plasmid described in any one of embodiments 49 to 87, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 14.

[0235] Embodiment 89: An rAAV vector described in any one of embodiments 11 to 48, 50 to 81, 87, or 88, or an rAAV vector plasmid described in any one of embodiments 49 to 88, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 15, or a functional subsequence, modification, or variant thereof.

[0236] Embodiment 90: An rAAV vector described in any one of embodiments 11 to 48, 50 to 81, or 87 to 89, or an rAAV vector plasmid described in any one of embodiments 49 to 89, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 15.

[0237] Embodiment 91: An rAAV vector described in any one of embodiments 11 to 48, 50 to 81, or 87 to 90, wherein the total length of the nucleic acid of the rAAV vector is approximately 5 kilobases or less in length, or 4.7 kilobases or less in length.

[0238] Embodiment 92: The rAAV vector plasmid of any one of embodiments 49 to 90, wherein the overall length of the rAAV vector plasmid is approximately 12 kilobases or less in length, or 4.7 kilobases or less in length.

[0239] Embodiment 93: A pharmaceutical composition comprising a nucleic acid molecule described in any one of embodiments 1 to 10, or an rAAV vector described in any one of embodiments 11 to 48, 50 to 81, or 87 to 91, and at least one pharmaceutically acceptable salt.

[0240] Embodiment 94: The pharmaceutical composition of embodiment 93, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 1 mM to about 450 mM.

[0241] Embodiment 95: The pharmaceutical composition of embodiment 93 or 94, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 2 mM to about 350 mM.

[0242] Embodiment 96: A pharmaceutical composition according to any one of embodiments 93 to 95, wherein the at least one pharmaceutically acceptable salt is sodium chloride, magnesium chloride, potassium chloride, calcium chloride, or calcium phosphate.

[0243] Embodiment 97: The pharmaceutical composition of embodiment 96, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and magnesium chloride.

[0244] Embodiment 98: The pharmaceutical composition of embodiment 96, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and potassium chloride.

[0245] Embodiment 99: A pharmaceutical composition according to any one of embodiments 93 to 98, further comprising at least one buffering agent.

[0246] Embodiment 100: The pharmaceutical composition of embodiment 99, wherein the buffering agent is citrate, histidine, acetate, phosphate, trishydrochloride, or tromethamine.

[0247] Embodiment 101: The pharmaceutical composition of embodiment 100, wherein the buffering agent is phosphate.

[0248] Embodiment 102: The pharmaceutical composition of embodiment 100, wherein the buffering agent is tris hydrochloride and tromethamine.

[0249] Embodiment 103: A pharmaceutical composition according to any one of embodiments 99 to 102, wherein the at least one buffering agent is present in an amount ranging from about 10 mM to about 40 mM.

[0250] Embodiment 104: The pharmaceutical composition of embodiment 103, wherein the at least one buffering agent is present in an amount of about 20 mM.

[0251] Embodiment 105: The pharmaceutical composition of any one of embodiments 93 to 104, further comprising at least one cryoprotectant.

[0252] Embodiment 106: The pharmaceutical composition of embodiment 105, wherein at least one cryoprotectant is a sugar or sugar alcohol.

[0253] Embodiment 107: The pharmaceutical composition of embodiment 105, wherein at least one cryoprotectant is trehalose, sucrose, sorbitol, or mannitol.

[0254] Embodiment 108: The pharmaceutical composition of embodiment 107, wherein at least one cryoprotectant is sucrose.

[0255] Embodiment 109: The pharmaceutical composition of embodiment 107, wherein at least one cryoprotectant is sorbitol.

[0256] Embodiment 110: A pharmaceutical composition according to any one of embodiments 105 to 109, wherein at least one cryoprotectant is present in an amount of up to about 20%.

[0257] Embodiment 111: The pharmaceutical composition of embodiment 110, wherein the at least one cryoprotectant is present in an amount ranging from about 3% to about 15%.

[0258] Embodiment 112: The pharmaceutical composition of embodiment 112, wherein the at least one cryoprotectant is present in an amount of about 4% or about 5%.

[0259] Embodiment 113: The pharmaceutical composition of any one of embodiments 93 to 112, further comprising at least one surfactant.

[0260] Embodiment 114: The pharmaceutical composition of embodiment 113, wherein at least one surfactant is a polaxamer or a polysorbate.

[0261] Embodiment 115: The pharmaceutical composition of embodiment 1114, wherein at least one surfactant is polaxamer 188, polysorbate 20, or polysorbate 80.

[0262] Embodiment 116: A pharmaceutical composition according to any one of embodiments 113 to 115, wherein the at least one surfactant is present in an amount ranging from about 0.0001% to about 1%.

[0263] Embodiment 117: The pharmaceutical composition of embodiment 116, wherein the at least one surfactant is present in an amount of about 0.02%.

[0264] Embodiment 118: The pharmaceutical composition of embodiment 116, wherein the at least one surfactant is present in an amount of about 0.002%.

[0265] Embodiment 119: A pharmaceutical composition according to any one of embodiments 93 to 118, wherein the pharmaceutical composition has a pH in the range of about 6 to about 8.

[0266] Embodiment 120: The pharmaceutical composition of embodiment 119, wherein the pharmaceutical composition has a pH in the range of about 7 to about 8.

[0267] Embodiment 121: The pharmaceutical composition of embodiment 120, wherein the pharmaceutical composition has a pH of about 7.6.

[0268] Embodiment 122: The pharmaceutical composition of embodiment 120, wherein the pharmaceutical composition has a pH of about 7.4.

[0269] Embodiment 123: A method for treating a heart-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of (a) a nucleic acid molecule according to any one of embodiments 1 to 10, or (b) an rAAV vector according to any one of embodiments 11 to 48, 50 to 81, or 87 to 91; or (c) A method comprising administering a pharmaceutical composition described in any one of embodiments 93 to 122.

[0270] Embodiment 124: The method of embodiment 123, wherein the heart-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

[0271] Embodiment 125: The method of embodiment 123 or 124, wherein the subject has a BAG3 mutation.

[0272] Embodiment 126: The method of any one of embodiments 123 to 125, wherein the heart-related disease or disorder is BAG3-associated dilated cardiomyopathy (DCM).

[0273] Embodiment 127: The method of any one of embodiments 123 to 125, wherein the heart-related disease or disorder is BAG3-associated heart failure.

[0274] Embodiment 128: The method of embodiment 123, wherein the heart-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

[0275] Embodiment 129: The method of embodiment 123 or 128, wherein the subject does not have a BAG mutation.

[0276] Embodiment 130: The method of any one of embodiments 123, 128, or 129, wherein the heart-related disease or disorder is heart failure unrelated to BAG3 expression.

[0277] Embodiment 131: A method for reducing the frequency or severity of at least one symptom associated with a heart-related disease or disorder in a subject, comprising administering to the subject: (a) a nucleic acid molecule according to any one of embodiments 1 to 10, or (b) an rAAV vector according to any one of embodiments 11 to 48, 50 to 81, or 87 to 91; or (c) A pharmaceutical composition according to any one of embodiments 93 to 122, administering in an amount effective to reduce the frequency or severity of at least one symptom.

[0278] Embodiment 132: The method of embodiment 131, wherein the heart-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

[0279] Embodiment 133: The method of embodiment 131 or 132, wherein the subject has a BAG3 mutation.

[0280] Embodiment 134: The method of any one of embodiments 131 to 133, wherein the heart-related disease or disorder is BAG3-associated dilated cardiomyopathy (DCM).

[0281] Embodiment 135: The method of any one of embodiments 131 to 133, wherein the heart-related disease or disorder is BAG3-associated heart failure.

[0282] Embodiment 136: The method of embodiment 131, wherein the heart-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

[0283] Embodiment 137: The method of embodiment 131 or 136, wherein the subject does not have a BAG mutation.

[0284] Embodiment 138: The method of any one of embodiments 131, 136, or 137, wherein the heart-related disease or disorder is heart failure unrelated to BAG3 expression.

[0285] Embodiment 139: The method of any one of embodiments 131 to 134, wherein at least one symptom is characteristic of BAG3-associated DCM.

[0286] Embodiment 140: The method of any one of embodiments 131 to 133 or 135, wherein at least one symptom is characteristic of BAG3-associated heart failure.

[0287] Embodiment 141: The method of any one of embodiments 131 or 136 to 138, wherein at least one symptom is characteristic of heart failure unrelated to BAG3 expression.

[0288] Embodiment 142: The method of any one of embodiments 123 to 141, wherein the effective amount of the rAAV vector is in the range of about 1E10 to about 1E17 vector genomes per kilogram of subject body weight (vg / kg).

[0289] Embodiment 143: The method of embodiment 142, wherein the effective amount of the rAAV vector is in the range of about 3E13 to about 1E14 vector genomes per kilogram of subject body weight (vg / kg).

[0290] Embodiment 144: The method of embodiment 142 or 143, wherein the effective amount of the rAAV vector is about 3E13 vector genomes per kilogram of body weight of the subject (vg / kg).

[0291] Embodiment 145: The method of embodiment 142 or 143, wherein the effective amount of the rAAV vector is about 7E13 vector genomes per kilogram of body weight of the subject (vg / kg).

[0292] Embodiment 146: The method of embodiment 142 or 143, wherein the effective amount of the rAAV vector is 1E14 vector genomes per kilogram of body weight of the subject (vg / kg).

[0293] Embodiment 147: Use of a nucleic acid molecule according to any one of embodiments 1 to 10 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0294] Embodiment 148: Use of an rAAV vector described in any one of embodiments 11 to 48, 50 to 81, or 87 to 91 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0295] Embodiment 149: Use of a pharmaceutical composition according to any one of embodiments 93 to 122 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

[0296] Embodiment 150: A plasmid comprising a nucleic acid molecule according to any one of embodiments 1 to 10.

[0297] Embodiment 151: A plasmid comprising an rAAV vector sequence described in any one of embodiments 11 to 48, 50 to 81, or 87 to 91.

[0298] Embodiment 152: A host cell for rAAV vector production, comprising the plasmid described in embodiment 150.

[0299] Embodiment 153: A host cell for rAAV vector production, comprising the plasmid described in embodiment 151.

[0300] Embodiment 154: A host cell for rAAV vector production, comprising an rAAV vector plasmid described in any one of embodiments 49 to 90.

[0301] Embodiment 155: The host cell according to any one of embodiments 152 to 154, wherein the host cell is a HEK293 cell or a derivative thereof.

[0302] Embodiment 156: The host cell of any one of embodiments 152 to 155, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep protein.

[0303] Embodiment 157: The host cell of any one of embodiments 152 to 156, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV9 capsid protein.

[0304] Embodiment 158: The host cell of any one of embodiments 152 to 157, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a viral helper factor.

[0305] Embodiment 159: A method of producing an rAAV vector, comprising: (a) incubating the host cell of any one of embodiments 152-158 under conditions sufficient to allow production of the rAAV vector; (b) purifying the rAAV vector produced thereby.

[0306] Embodiment 160: A rAAV vector produced by the method of embodiment 159.

[0307] [Table 1-1]

[0308] [Table 1-2]

[0309] [Table 1-3]

[0310] Table 1-4

[0311] Table 1-5

[0312] Table 1-6

[0313] Table 1-7

[0314] Table 1-8

[0315] Table 1-9

[0316] Table 1-10

[0317] Table 1-11

[0318] Table 1-12

[0319] Table 1-13

[0320] [Table 1-14]

[0321] [Table 1-15] [Example]

[0322] The following examples are intended for illustrative purposes only and are not meant to limit the present disclosure in any way.

[0323] Example 1: AAV Vector Production HEK293 cells were grown in suspension culture and transfected with three plasmids using standard methods known in the art to produce rAAV vectors. One of the plasmids contained Compound A. HEK293 cells were harvested, lysed, and aggregated, and the resulting lysate was filtered to produce a clarified lysate. The rAAV vector containing the BAG3 transgene was purified through a series of chromatography and filtration steps and suspended in a formulation containing 350 mM NaCl, 2.7 mM KCl, 5% sorbitol, and 10 mM phosphate at pH 7.4 with 0.002% Pluronic.

[0324] [Table 2]

[0325] [Table 3]

[0326] [Table 4]

[0327] Example 2: Pharmacology Compound A pharmacology was evaluated in vitro and in vivo. Cell-based assay data demonstrated that Compound A effectively transduced and induced the synthesis of functional BAG3 protein in wild-type human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs). Human BAG3 protein expression was detected in the hearts of wild-type (WT) mice and WT non-human primates (NHPs) after Compound A administration. Compound A administration in a mouse disease model prevented further deterioration of the disease process. Specifically, it stabilized cardiac structure and function, which correlated with the dose level and amount of human BAG3 protein expression in the heart. Compound A vector transduction and human BAG3 protein expression were evaluated in NHPs. Compound A-treated NHPs maintained human BAG3 expression in the heart at the predicted efficacious therapeutic target level.

[0328] [Table 5]

[0329] In vitro primary pharmacology Compound A was tested in wild-type human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs, CDI iCell Cardiomyocytes) to demonstrate transduction (viral genome count, VGC), transgene expression (mRNA, protein), and stabilization of the BAG3 partner protein (HSPB8). HiPSC-CMs were used to perform efficacy assays in a cell type in which the cardiac troponin promoter element is active, thus allowing transgene expression. HSPB8 is a BAG3 partner protein whose stability depends on the presence of functional BAG3 and has been used as a surrogate for BAG3 activity (Fang et al., JCI Insight 4(4):e126464 (2019); Fang et al., J. Clin. Invest. 127(8):3189-200 (2017); Judge et al., JCI Insight 2(14):e94623 (2017)).

[0330] HiPSC-CMs were transduced with Compound A, and transgene mRNA was readily detected 4 days after transduction (Figure 1). Total BAG3 protein (human endogenous + human transgene) was measured using the Protein Simple WES platform. Total BAG3 protein levels showed a dose-responsive increase with increasing vector multiplicity of infection (MOI).

[0331] HiPSC-CMs were first transfected with siRNA targeting endogenous BAG3 expression, which resulted in significant knockdown of endogenous BAG3 protein expression and HSPB8 destabilization (Figure 2). Subsequently, HiPSC-CMs were transduced with Compound A, resulting in robust upregulation of total BAG3 protein (human endogenous + human transgene) and stabilization of the HSPB8 partner protein.

[0332] In vivo primary pharmacology Primary in vivo pharmacology was investigated in wild-type mice (biodistribution and expression), in mouse models of disease (model characterization and dose-response related efficacy assessment), and in non-human primates (biodistribution and expression).

[0333] Assessment of biodistribution and expression in wild-type mice Wild-type mice (n: 12, age: 8 weeks) were administered Compound A (3E13vg / kg) and autopsies were performed 3 and 8 weeks after administration. No significant difference in body weight was detected between Compound A-treated and vehicle-treated control mice during the study (Figure 3A).

[0334] Viral genome copies were readily detected in the hearts of Compound A-treated mice at 3 and 8 weeks post-administration (Figure 3B). At 8 weeks, liver viral genome levels were approximately 175-fold higher than those in the heart. Transgene mRNA expression was highest in the heart and at similar levels at both time points. In the liver, transgene mRNA expression was approximately 40-50% of the signal in the heart (Figure 3C). Transgene protein expression (assayed using an antibody specific for the human BAG3 protein) was readily detected in the hearts of Compound A-treated mice, but no signal was detected in skeletal muscle and a low signal was detected in the liver (Figure 3D). Quantification of total BAG3 protein in the heart revealed similar levels of BAG3 expression in the hearts of Compound A-treated mice as in control vehicle-treated mice.

[0335] Cardiac function (left ventricular ejection fraction) and structure (left ventricular end-diastolic volume) were measured by echocardiography at 4 and 7 weeks after administration. No significant differences were detected in treated compared to vehicle-treated control mice (Figure 3E). Finally, the percentage of area in the heart expressing the human transgene was assayed using BAG3 immunohistochemistry and showed similar biodistribution at 3 and 8 weeks (74% and 56%, respectively) (Figure 3F).

[0336] Example 3: Characterization of cKO mouse models of disease: Mice with cardiac-specific knockdown of BAG3 (BAG3 cKO, n:20, male and female) (Fang et al., J. Clin. Invest. 127(8):3189-200 (2017)) and wild-type control mice (BAG3 cWT, n:20, male and female) were longitudinally characterized using echocardiography between 7 and 21 weeks of age (Figure 4A-B). A small but significant defect in ejection fraction was detected as early as 7-8 weeks of age (BAG3 cKO: 64.2 ± 6.0% vs. BAG3 cWT: 70.5 ± 3.9%), which became significant by 18-21 weeks of age (BAG3 cKO: 31.9 ± 8.7% vs. BAG3 cWT: 72.2 ± 3.6%). Similarly, a significant increase in left ventricular end-diastolic volume was observed in BAG3 cKO mice compared to wild-type controls at 18–21 weeks of age (BAG3 cKO: 93.4 ± 30.4% vs. BAG3 cWT: 61.5 ± 9.0%). No significant differences in body weight were detected between the two cohorts between 11–15 weeks of age and 21–23 weeks of age (Figure 4C).

[0337] As expected, BAG3 protein (measured by Protein Simple WES assay) was absent in the hearts of cKO mice, but it was readily detected in the hearts of control wild-type mice (Figure 4D). A significant decrease in HSPB8 protein was observed in the hearts of BAG3 cKO mice, with the signal being approximately 20% of that measured in wild-type control mice (Figure 4E). BAG3 protein levels in the liver and skeletal muscle tissues of BAG3 cKO mice were similar to those of wild-type mice.

[0338] Expression levels (mRNA) of biomarkers of heart failure (Nppa, Nppb, Myh7 / Myh6 ratio) and fibrosis (Col1a1, Col1a2, Fn1, Postn, Timp1) were significantly upregulated in the hearts of BAG3 cKO mice compared to wild-type control mice (Figure 4F-G). Finally, based on histological evaluation of cardiac tissue (picrosirius red staining), a significantly higher rate of fibrosis was observed in the hearts of BAG3 cKO mice compared to control wild-type mice (Figure 4H).

[0339] Example 4: Evaluation of dose-response related efficacy in mouse models of disease: To determine whether restoring BAG3 expression in the heart via AAV gene delivery would result in a therapeutic effect, a dose-response efficacy study was conducted in a cKO mouse model of disease. BAG3 cKO mice were administered Compound A at 1E13vg / kg (n:15), 3E13vg / kg (n:15), or 9E13vg / kg (n:15) IV (age: 12 weeks). The study also included two control groups: untreated BAG3 cKO mice (n:20) and untreated wild-type mice (n:20). Two weeks prior to treatment, mice were characterized using echocardiography to determine baseline cardiac function and structure. Mice were randomly assigned to groups based on echocardiographic readings (ejection fraction, left ventricular end-diastolic volume), body weight, and sex.

[0340] Cardiac function (ejection fraction) and structure (left ventricular end-diastolic volume) were measured longitudinally using echocardiography at 4, 8, and 12 weeks after administration (Figure 5A-B). At week 12, significant improvements in ejection fraction were detected in mice treated with Compound A at 3E13 vg / kg (42.5 ± 11.4%) and 9E13 vg / kg (47.9 ± 7.3%) compared to untreated BAG3 cKO mice (28.2 ± 10.0%). At week 12, significant improvements in left ventricular end-diastolic volume were detected in mice treated with Compound A at 3E13 vg / kg (78.5 ± 12.2 μL) and 9E13 vg / kg (71.7 ± 9.0%) compared to untreated BAG3 cKO mice (97.2 ± 32.5 μL). No significant differences in body weight were detected between these mouse groups (Figure 5C).

[0341] Viral genome biodistribution was investigated in heart, liver, and skeletal muscle tissues collected at necropsy from mice treated with Compound A at 1E13vg / kg, 3E13vg / kg, and 9E13vg / kg (Figure 5D). A clear dose response in biodistribution was detected in the heart: 1E13vg / kg (0.03 ± 0.01vg / mTFRC), 3E13vg / kg (0.19 ± 0.1vg / mTFRC), and 9E13vg / kg (0.6 ± 0.2vg / mTFRC). The highest viral genomes were detected in the liver (40-45-fold higher than in the heart), while lower vector genomes were observed in skeletal muscle (0.2-fold lower than in the heart). Transgene mRNA expression was highest in the heart compared to other tissues investigated, with the liver having approximately 0.1-fold higher than in the heart, and skeletal muscle having approximately 0.01-fold higher than in the heart (Figure 5E).

[0342] Total BAG3 protein expression (human + mouse) was determined using a Protein Simple WES assay. BAG3 protein was not detected in the hearts of untreated control cKO mice. A dose-responsive increase in BAG3 protein levels was observed in cKO mouse hearts after treatment with Compound A. At the highest dose, approximately 80% (0.78 ± 0.45) of WT BAG3 levels was detected, and at the mid-dose, approximately 20% (0.20 ± 0.11%) of WT BAG3 levels were observed (Figure 5F). Compound A also induced a dose-responsive increase in HSPB8 protein expression in the same cardiac tissue samples: approximately 70% (0.68 ± 0.20) at the highest dose and approximately 35% (0.34 ± 0.06%) of wild-type levels at the mid-dose (Figure 5G).

[0343] Human BAG3 protein expression was quantified using an LC / MS assay specific for the human sequence: at 9E13vg / kg, expression was approximately 70% of the level in wild-type mouse hearts, and at 3E13vg / kg, it was approximately 20% of the level expressed in wild-type mouse hearts (Figure 5H). Spatial expression of the transgene was determined using in situ hybridization (human BAG3 mRNA) and immunohistochemistry (BAG3 protein, antibody specific for the human protein) (Figures 5I-5J). At the 9E13vg / kg dose, 52±18% of cardiomyocytes were positive for human BAG3 mRNA, and 52±14% of the cardiac region were positive for BAG3 protein. At the 3E13vg / kg dose, 39±26% of cardiomyocytes were positive for transgene mRNA, and 29±15% of the cardiac region were positive for transgene protein.

[0344] Biomarkers of heart failure and fibrosis were investigated using RT-PCR (Figure 5K). Compared with untreated control mice, treatment with 9E13 and 3E13 vg / kg of Compound A resulted in significant decreases in Nppa, Nppb, and the Myh7 / Myh6 ratio. Significant decreases were also detected in the levels of Col1a1 and Postn expression, but no significant changes were detected in Col1a2, Fn, and Timp1 levels. No significant differences were detected in NT-proBNP levels measured in serum after treatment.

[0345] Example 5: Evaluation of biodistribution and expression in non-human primates: To determine the extent of viral genome biodistribution in non-human primates, cohorts of animals were treated with 3E13 and 1E14 doses of Compound A. Repeated titrations after administration provided a more accurate titer of the administered vector, resulting in actual dose levels of 4E13 and 1.3E14 vg / kg. The actual dose levels were used for modeling and dose prediction in Example 8. Each dose level was administered to one male and one female adult animal. The study also included a pair of vehicle-treated animals. The vehicle consisted of 0.002% Pluronic F68 (pH 7.4) in 10 mM phosphate, 350 mM NaCl, 2.7 mM KCl, and 5% sorbitol in sterile water for injection. The active period of the study was 6 weeks long.

[0346] The number of viral genomes detected in the heart increased with vector dose: 0.86 ± 0.15 vg / MfTFRC (4E13 vg / kg) and 2.72 vg / MfTFRC (1.3E14 vg / kg) (Figure 6A). Viral genomes in the liver were approximately 167-243 times higher than those in the heart. Viral genomes were also detected in the dorsal root ganglia (0.12-0.35 times the heart level), spinal cord (0.06 times the heart level), skeletal muscle (0.2-0.35 times the heart level), and testes or ovaries (0.04-0.08 times the heart level). One female animal administered the 1.3E14 vg / kg dose showed very low biodistribution, which was determined to have seroconverted and possessed neutralizing antibodies to AAV9 prior to administration.

[0347] Transgene mRNA expression levels were quantified and normalized to either endogenous HPRT or endogenous BAG3 (Figures 6B-6C). Expression levels achieved in the heart were approximately 6-8-fold higher in animals treated with the 1.3E14vg / kg dose compared to the 4E13vg / kg dose. Transgene mRNA expression in the liver was higher than that detected in the heart: 1.8-fold at the 4E13vg / kg dose and 6.4-fold at the 1.3E14vg / kg dose. Transgene expression in the dorsal root ganglia was approximately 0.44-fold higher than that detected in the heart at the 1.3E14vg / kg dose. Much lower signals were detected in the spinal cord and skeletal muscle. Normalization of transgenic BAG3 mRNA levels to endogenous BAG3 levels revealed that in the heart, at the 4E13vg / kg dose level, transgene mRNA levels were approximately 0.1-fold higher than endogenous BAG3 levels, and at the 1.3E14vg / kg dose level, 0.8-fold higher. In the liver, the signal was higher: 3-fold higher than endogenous at 4E13vg / kg and 25-fold higher than endogenous at 1.3E14vg / kg.

[0348] Human BAG3 protein expression was measured using LC / MS (Figures 6E-6F). A peptide with a conserved sequence in both human and cynomolgus monkey BAG3 proteins was used to measure total protein, and a peptide with a sequence specific to the human protein was used to quantify human protein. Comparing BAG3 levels in the heart, 369 ng / mg protein was detected at the 1.3E14 vg / kg dose, 248 ng / mg protein at the 4E13 vg / kg dose, and 189 ng / mg protein in vehicle-treated animals.

[0349] In non-cardiac tissues of vehicle-treated animals, endogenous BAG3 levels were highest in the spinal cord (157 ng / mg) and lowest in the liver (7 ng / mg). Human transgene protein was detected only in animals administered Compound A. At a dose of 4E13 vg / kg, approximately 13.9 ng / mg (male animals) and 4.6 ng / mg (female animals) of BAG3 protein were detected in the heart, but no transgene protein was detected in the liver, dorsal root ganglia, spinal cord, or skeletal muscle. At a dose of 1.3E14 vg / kg, approximately 70 ng / mg of BAG3 protein was detected in the heart, but no transgene protein was detected in the dorsal root ganglia, spinal cord, or skeletal muscle. A small signal (approximately 3 ng / mg) was detected in the liver. Compared to total BAG3 levels, human BAG3 protein levels detected in the heart were approximately 4% of total BAG3 levels at the 4E13vg / kg dose level and approximately 30% of total BAG3 at 1.3E14vg / kg.

[0350] Spatial BAG3 expression was assessed in the heart using in situ hybridization (Figure 6D). At the 4E13vg / kg dose, 30±11% (female) and 64±19% (male) of cardiomyocytes stained positive by ISH. At the 1.3E14vg / kg dose, 89±5% of cardiomyocytes stained positive by ISH (male animals; seroconverted female animals were excluded from this analysis).

[0351] Example 6: Toxicology and Safety Pharmacology overview The nonclinical safety of Compound A was evaluated in both WT and cKO mice (pharmacology) using biodistribution and toxicity endpoints. Additionally, a 6-week exploratory toxicology and biodistribution study (ETS) in cynomolgus monkeys was completed. Safety pharmacology endpoints or the potential for insertional mutagenesis were not evaluated in these initial studies.

[0352] In the WT mouse study, animals were administered Compound A at 3E13 vg / kg by IV bolus with a limited list of biodistribution, clinical pathology, and histopathology endpoints evaluated at 3 and / or 8 weeks post-dose. There was target expression in WT mice at 3E13 vg / kg with no evidence of toxicity.

[0353] In the cKO mouse study (1E13, 3E13, and 9E13 vg / kg Compound A), histopathological findings were limited to high-dose animals evaluated 12 weeks after dose administration. Findings associated with Compound A at the high dose (9E13 vg / kg) consisted of mild cardiomyocyte degeneration / necrosis; however, these were not associated with changes in cardiac biomarker endpoints of toxicity and occurred in the presence of functional efficacy (i.e., echocardiographic improvement). Functional improvement in the disease mouse model was identified at doses ≥3E13 vg / kg, identifying the mouse efficacy range.

[0354] In a 6-week monkey ETS, Compound A was well tolerated at both dose levels evaluated (3E13 and 1E14 vg / kg), with no Compound A-related microscopic findings. Repeat titrations after administration provided a more accurate titer of the administered vector, with actual dose levels of 4E13 and 1.3E14 vg / kg. Based on transient clinical pathology changes (increased ALT and GLDH, ≥4E13 vg / kg, increased troponin at 4E13 vg / kg) and increased cytokines (TNF at 1.3E14 vg / kg) in the presence of daily immunosuppressive regimens, the heart, liver, and immune system may be potential target organs with higher doses and / or longer durations.

[0355] In summary, early nonclinical toxicology evaluations in mice (WT and cKO) and monkeys provided an adequate safety profile for continued development and supported dose selection for evaluation in regulatory toxicology.

[0356] Toxicology Species Selection The cynomolgus monkey (cyno) was selected as the relevant toxicology species for evaluation of Compound A. Cynomolgus monkeys were selected based on the following weight of evidence: (i) high target homology, (ii) ability to support transgene expression, (iii) demonstrated transgene protein localization at the site of action, and (iv) species sensitivity for toxicity assessments relevant to gene therapy, such as acute liver toxicity and microscopic changes in the dorsal root ganglion.

[0357] BAG3 shares approximately 97% identity across the full-length protein sequence between cynomolgus monkeys and humans. Furthermore, the substantial similarity between cynomolgus monkey and human BAG3 indicates that all relevant binding partners and interactions are likely to be maintained. BAG3 primarily binds to partner proteins via its WW1, WW2, IPV1, IPV2, PXXP, and BAG domains. Comparison of the cynomolgus monkey and human BAG3 protein sequences reveals that the WW1, IPV1, IPV2, and BAG domains share identical sequences, while the WW2 and PXXP domains share 97% and 92% identity. This finding is further supported by the results of a cynomolgus monkey ETS study, which demonstrated significant and dose-responsive human BAG3 expression by in situ hybridization (BAG3 mRNA) in cardiac tissue using digital imaging analysis. Importantly, human BAG3 expression in cynomolgus monkeys led to detectable and dose-responsive human BAG3 protein levels in the heart.

[0358] Furthermore, LCMS evaluation of purified sarcomeres isolated from cynomolgus monkey ETS hearts (three heart sections / animal) identified a single peptide unique to the human isoform. This peptide showed a dose-dependent signal increase across two dose levels of the administered human BAG3 transgene (Figure 7), confirming the localization of human BAG3 at the sarcomere site of action. Finally, the human BAG3 transgene was found to rescue cardiac function defects in BAG3 cKO mice, despite sequence differences between human and mouse BAG3 (84% identity). Taken together, BAG3 homology, expression, and localization support the relevance of cynomolgus monkeys as a toxicology species, along with species sensitivity for toxicity assessment related to gene therapy.

[0359] Three exploratory toxicity (eTox) studies conducted 1. Exploratory WT mouse biodistribution and tolerability study at 3 and 8 weeks following a single IV bolus of Compound A at 3E13vg / kg. For biodistribution and toxicity studies, male wild-type (WT) mice (BL6 / J; 8 weeks old) were administered Compound A intravenously via tail vein injection at 3E13 vg / kg and necropsied 3 or 8 weeks after injection. A separate control group received a single saline injection. End-point assessments of the heart, liver, and skeletal muscle included molecular biology (VGC, RNA, protein; shown in Figures 3B-3D), clinical pathology, and histopathology, as well as IHC (protein; Figure 3F; Figure 8), and ISH (RNA; Figure 9). A targeted panel of clinical pathology parameters consisted of liver, heart, and skeletal muscle biomarkers (ALT, AST, ALP, total bilirubin, GLDH, cardiac troponin (cTNI), fatty acid binding protein 3 (FABP3), myosin light chain 3 (Myl3), and skeletal troponin I (sTnI)). Animals were evaluated for potential Compound A-related microscopic tissue effects (heart, liver, skeletal muscle, brain, adrenal glands, kidneys, lungs, spinal cord, and dorsal root ganglia [DRG]) 8 weeks after dose administration.

[0360] [Table 6]

[0361] All mice survived until scheduled sacrifice at 3 or 8 weeks, and there were no Compound A-related changes in body weight (Figure 3A), clinical pathology markers of the heart (cTnI, Myl3, FABP3), clinical pathology markers of the liver (ALT, AST, ALP, total bilirubin, GLDH), or clinical pathology markers of skeletal muscle injury (sTnI, Myl3, FABP3) compared to controls. No Compound A-related histopathological findings were observed at 8 weeks.

[0362] Human BAG3 protein immunoreactivity was assessed by IHC in the heart, liver, and skeletal muscle. IHC revealed a broad expression pattern with variable intensity across animals administered 3E13 vg / kg of Compound A. The BAG3 cardiac expression pattern and intensity were generally similar at 3 or 8 weeks, with the cardiac region staining (Figure 3F; Figure 8). The left ventricular free wall and interventricular septum of the heart showed slightly greater intensity compared to other locations. Very low expression of BAG3 protein by IHC was observed in the liver of all animals in the centrilobular region, as well as minimal expression in the DRG. No BAG3 expression was observed in skeletal muscle.

[0363] Human BAG3 ISH (mRNA) was completed in the heart, liver, and skeletal muscle from selected animals and in DRG from all animals at 8 weeks. Representative images are shown in Figure 9. ISH labeling in the heart was nuclear and cytoplasmic, variable, and labeled fewer cardiomyocytes than IHC. In the liver, ISH labeling was mostly nuclear and frequently observed in hepatocytes. There was minimal human BAG3 RNA expression in the DRG. Skeletal muscle was BAG3 negative.

[0364] In summary, Compound A administered by its intended route in the clinic at 3E13 vg / kg was well tolerated in WT mice at 3 and 8 weeks after IV administration. No Compound A-related clinical pathology changes were observed in markers of cardiac, liver, or skeletal muscle injury or microscopic findings. IHC evaluation revealed a broad and variable pattern of BAG3 human protein expression in the heart of all animals, whereas there was very low level expression in the liver and DRG, and no expression was observed in skeletal muscle.

[0365] 2. Exploratory cKO mouse biodistribution and tolerability study (12 weeks) following a single IV bolus of Compound A at 1E13, 3E13, and 9E13 vg / kg. Exploratory pharmacology / toxicology studies were conducted in 12-week-old BAG3 cKO dilated cardiomyopathy (DCM) mice treated with a single intravenous tail vein injection of saline or Compound A. Necropsies were performed 12 weeks after administration of a single dose of Compound A at 1E13, 3E13, or 9E13 vg / kg. Cohorts of cKO and WT mice were included to establish a baseline for comparison at the start of the study.

[0366] [Table 7]

[0367] [Table 8]

[0368] Mortality occurred across treatment groups, including cKO saline control animals (Table 8 above), and was attributed to DCM disease and not related to Compound A treatment. Necropsies were performed approximately 12 weeks post-dose, and serum was collected. No Compound A-related differences in a targeted panel of clinical pathology parameters were observed across groups (ALT, AST, ALP, GLDH, TBIL, cTNI, MYL3, sTNI, and FABP3). NT-proBNP (indicating myocyte elongation), a marker of cardiotoxicity, was found to be elevated in the cKO mouse control group at 12 weeks post-dose compared to WT mice, indicating pathological progression of the disease model. There were no Compound A-related changes in NT-proBNP at 12 weeks post-dose.

[0369] Tissues collected 12 weeks post-treatment included heart, liver, skeletal muscle, spinal cord / DRG, lung, kidney, adrenal gland, and brain. However, only heart, liver, and skeletal muscle were evaluated for potential histopathological findings. Microscopic cardiac evaluation of untreated control cKO mice at baseline (11–12 weeks of age) showed minimally severe microscopic changes consistent with the DCM disease model (Table 9; severity scores compared to WT mice). Saline control cKO hearts at the end of the study (23–25 weeks of age) were similar to the baseline cohort of cKO mouse hearts, except for findings of mild to moderate ventricular dilation and mild cardiomyocyte atrophy.

[0370] In the cKO group treated with 3E13vg / kg Compound A, signs of efficacy were seen by functional assessment (echocardiography, Figures 5A and 5B) and microscopic assessment (reduced mean severity of ventricular dilatation and cardiomyocyte atrophy). This dose level (3E13vg / kg) had no toxicity findings related to Compound A, although rare hyperkaryotic cardiomyocytes with cytoplasmic basophilia were observed. The cKO group treated with the higher dose (9E13vg / kg) of Compound A also showed functional improvement (Figures 5A and 5B) and microscopic signs of efficacy in the heart. Efficacy was still evident at the higher dose, but Compound A-associated cardiomyocyte degeneration / necrosis was mild, with karyomegaly, cytoplasmic basophilia, and occasional vacuolization. These microscopic findings in the high-dose animals occurred in the presence of functional (efficacy) outcomes without changes in biomarkers of cardiac toxicity, indicating continued improvement. Therefore, clinical dosing into this range (ie, 9E13 vg / kg) is not excluded.

[0371] Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to assess the biodistribution of transgene expression in heart, liver, and skeletal muscle (other tissues, including spinal cord / DRG, were not assessed). Human BAG3 protein immunoreactivity by IHC generally increased with Compound A dose, with variable distribution of signal observed within treatment groups and within individual heart samples (Figure 10). Digital image analysis showed that an average of 52% of hearts were positive for BAG3 protein (Table 9; Figure 5J). Human BAG3 mRNA was detected by ISH, and the signal was also observed to increase with Compound A dose. Digital image analysis showed that up to 52% of cardiomyocytes were positive for human BAG3 expression across dose groups (Table 9; Figure 10; Figure 5I).

[0372] [Table 9]

[0373] In summary, evaluation of Compound A (1E13, 3E13, 9E13 vg / kg) in BAG3 cKO mice administered via the intended clinical route of administration demonstrated functional improvement at doses ≥3E13 vg / kg, as well as dose-dependent expression by ISH (RNA) and IHC (protein) in cardiomyocytes. Evaluation of a targeted set of safety endpoints 12 weeks after administration did not reveal any Compound A-related clinical pathology changes in markers of liver, cardiac, or skeletal muscle toxicity. At the high dose of 9E13 vg / kg (approximately three-fold higher than the effective dose of 3E13 vg / kg in this study), mild cardiomyocyte degeneration / necrosis associated with Compound A was observed. These microscopic findings occurred in the presence of continued functional (efficacy) improvement without changes in biomarkers of cardiac toxicity.

[0374] 3. Single-dose IV exploratory toxicity and biodistribution study in cynomolgus monkeys (6 weeks) at 4E13 and 1.3E14 vg / kg of Compound A. Male and female cynomolgus monkeys, approximately 3-4 years old at the start of dosing, received a single dose of either vehicle or Compound A via IV injection. The purpose of this study was to investigate the potential toxicological effects and biodistribution in cynomolgus monkeys after a single IV dose of Compound A at 3E13 and 1E14 vg / kg. It is important to note that based on a re-potency analysis of the lots completed after the study, the stock concentration was revised from 5.02E13 vg / mL to 6.7E13 vg / mL, resulting in the intended doses of 3E13 and 1E14 vg / kg actually being 4E13 vg / kg and 1.3E14 vg / kg. All groups received methylprednisolone at 5 mg / kg once daily via IM injection, starting the day before and continuing until the day before necropsy.

[0375] [Table 10]

[0376] Animals were prescreened for anti-AAV9 neutralizing antibodies (nAbs) prior to dosing, and negative animals were selected for testing. nAb evaluation used a cell-based transduction inhibition assay. nAb titers were reported as the reciprocal of the lowest serum dilution (5, 10, 20, 40) that had ≥50% transduction inhibition. Samples with titers <5 were considered nAb-negative for AAV9. However, upon rechecking after dosing, it was confirmed that the high-dose female (1.3E14 vg / kg) seroconverted with a high AAV9 nAb titer, explaining the lack of target protein expression and affected immune endpoint evaluation observed in this animal.

[0377] All animals survived to scheduled necropsy without clinical observations related to Compound A. There were no gross or microscopic findings related to Compound A.

[0378] At ≥ 4E13vg / kg, Compound A-related increases were observed in ALT, AST, GLDH, CK, cardiac troponin I, neutrophils, and monocytes.

[0379] At 4E13 vg / kg, ALT increased on days 3 (females) and 8 (males and females) and returned to baseline by day 15 (Figure 11A). At 1.3E14 vg / kg, ALT increased (peak) on day 15 and remained above baseline until day 43 in males only. AST increased at 4E13 vg / kg on day 3 (females) or 1.3E14 vg / kg on day 8 (males) and returned to baseline by day 15. GLDH also transiently increased at 1.3E14 vg / kg on days 8 (peak) and 15 in males and at 4E13 vg / kg on days 3 (peak) and 8 in females. The increases in ALT and GLDH indicate hepatocellular injury, but there was no microscopic correlation from histopathological evaluations performed on liver samples (necropsy on days 44 or 47). In males only, at 1.3E14 vg / kg, CK was increased on days 3 (peak) and 15, returning to baseline by day 29. Cardiac troponin I was elevated in female animals only at 4E13 vg / kg on day 43, consistent with the upward trend at the end of the study. In males only at 1.3E14 vg / kg, neutrophils, monocytes, and LUC were increased on days 3, 8, and / or 43.

[0380] Evaluation of cytokines and complement factors revealed Compound A-related changes at the high dose. There were no Compound A-related changes in cytokines or complement activation products in animals administered 4E13 vg / kg Compound A. At 1.3E14 vg / kg, an increase in TNF (Figure 11B) was observed in males (7.6-fold baseline; day 15) and females (4.8-9.4-fold baseline; peak at day 15), and SC5b-9 was increased (53-fold baseline; day 1 at 6 HPD). Complement activation (SC5b-9) in high-dose females (1.3E14 vg / kg) was likely initiated by the AAV9 nAb and was not believed to be directly related to Compound A administration. Together with complement activation, the AAV9 nAb likely enhanced the earlier onset and greater magnitude of the TNF increase observed in high-dose females compared to high-dose males. The nAb responses from the pre-dose sample revealed that this animal had seroconverted prior to dosing, indicating that the animal was no longer immunologically naive, potentially affecting subsequent immune endpoint assessments. A positive AAV9 capsid ELISpot response was observed in this high-dose animal and was likely a recall / memory cell-mediated response as a result of previous AAV9 exposure, as evidenced by seroconversion prior to Compound A dosing. There were smaller, more equivocal ELISpot signals observed in males at both dose levels that were not considered related to Compound A dosing.

[0381] The biodistribution (VGC, RNA, BAG3 protein) of Compound A at 4E13vg / kg and 1.3E14vg / kg was assessed using selected tissues shown in Table 11 below (see also Figures 6A-6F). Cardiac expression was observed at ≥4E13vg / kg, accompanied by protein translation, in all animals except high-dose females, which were due to seroconversion. Evidence of expression was observed in the liver (protein in males only) and DRG neurons (no protein observed) without histopathological findings.

[0382] [Table 11]

[0383] In situ hybridization for BAG3 RNA was performed on cardiac sections from males and females in all groups. Positive signals, present as punctate red dots (Figure 12), were present in the nuclei and cytoplasm of cardiomyocytes in all animals treated with Compound A, except for high-dose females. The intensity and extent of positive ISH labeling in cardiomyocytes varied across cardiac samples and individual animals. Generally, labeling was higher (number of red dots per cardiomyocyte and / or number of cardiomyocytes with red dots) with increasing dose in males and was higher in males than in females at 4E13vg / kg. In addition to semiquantitative assessment of the percentage of positive cardiomyocytes by a pathologist, digital image analysis was performed to quantify the percentage of positive cardiomyocytes (Figure 6D). There was moderate variability in positive labeling between cardiac sections from a given animal within a group. The percentage of BAG3 ISH-positive cardiomyocytes in individual sections in males and females treated with 4E13vg / kg ranged from 45 to 92% and 20 to 56%, respectively. In males administered 1.3E14vg / kg, these values ​​ranged from 79 to 95%.

[0384] In summary, Compound A administered via the intended clinical route of administration at 4E13 and 1.3E14 vg / kg was well tolerated in monkeys in the presence of a daily IS regimen for 6 weeks after IV infusion. There were no histopathological findings associated with Compound A. Clinical pathology results showed increases in ALT, AST, CK, cardiac troponin I, neutrophils, and monocytes associated with Compound A at doses ≥4E13 vg / kg. In addition, at the higher dose (1.3E14 vg / kg), there were changes in immune endpoints (cytokines, complement, ELISpot), and one high-dose animal was seroconverted prior to dosing. Cardiac in situ hybridization for BAG3 RNA demonstrated a high percentage of BAG3-positive cardiomyocytes across cardiac sections in high-dose males, ranging from 20% to 92% at 4E13 vg / kg and from 79% to 95% at 1.3E14 vg / kg.

[0385] Example 7: Tissue Distribution / Tropism BAG3 biodistribution was assessed in cynomolgus monkey ETS after IV administration of Compound A up to 1.3E14 vg / kg (approximately 3.3-fold the efficacious dose demonstrated in cKO mice) (see Table 11 and Figures 6A-6F). Viral genome copies (VGCs) were present in the heart, liver, skeletal muscle, gonads, spinal cord, and DRG, generally increasing in a dose-dependent manner (Figure 6A). There was evidence of VGCs in these tissues, but no histopathological findings. AAV9 vector-mediated BAG3 expression (RNA) was detected primarily in the liver and heart (Figure 6C), although expression was also observed in skeletal muscle, spinal cord, and DRG (gonads were not assessed for RNA). ISH demonstrated significant and dose-dependent BAG3 expression throughout cynomolgus monkey cardiomyocytes (Figure 6D). Total BAG3 (conserved) protein was found in the heart, liver, skeletal muscle, spinal cord, and DRG of cynomolgus monkeys, whereas the human-specific peptide confirmed that human BAG3 protein was primarily localized in the heart at both dose levels evaluated. Little human BAG3 protein was detected in the liver at high doses, and no protein was detected in the spinal cord or DRG (Figures 6E-F).

[0386] Example 8: Pharmacokinetic-pharmacodynamic relationships and prediction of effective human doses. The dose-conversion plan for Compound A involved two steps: First, the effective level of target (BAG3) expression and cardiac coverage was determined using data from a disease model mouse (BAG3-cKO) dose-response study. Second, the dose required to achieve effective BAG3 expression in a mouse-based setting was determined using data from a cynomolgus monkey study in which two different dose levels of Compound A were tested to evaluate the dose-response of BAG3 expression in the monkey heart.

[0387] This translational plan assumed that the BAG3 expression (mRNA and protein)-efficacy relationship in the BAG3 cKO mouse disease model would represent the same relationship in BAG3 DCM patients. Additionally, we hypothesized that the dose-BAG3 expression relationship in humans could be based on the dose-BAG3 relationship observed in cynomolgus monkeys. These assumptions and data from the BAG3 cKO mouse and cynomolgus monkey studies were integrated into a quantitative systems pharmacology model for AAV gene therapy (developed and validated using available literature and in-house data) to generate effective dose and dose-dependent BAG3 expression predictions, as described below. Note that due to the small number of animals in the cynomolgus monkey ETS study used to plan the clinical dose (n = 2 at 4E13vg / kg and n = 1 at 1.3E14vg / kg), dose predictions may be updated with data from future studies.

[0388] Preclinical pharmacological and therapeutic levels of BAG3 expression Target therapeutic levels of BAG3 expression in the heart were determined based on results from a dose-response efficacy study in the BAG3 cKO mouse model of disease described above. Echocardiographic changes in cardiac structure and function after treatment in individual animals observed in this study were correlated with corresponding BAG3 mRNA and protein expression measured in cardiac tissue at necropsy to determine the BAG3 expression-efficacy relationship (Figures 13A-D).

[0389] Specifically, human BAG3 protein expression in BAG3 cKO mouse hearts (quantified using an LC / MS assay specific for the human BAG3 protein) increased wild-type mouse cardiac BAG3 expression from 0 (control) to 70% (9E13vg / kg) with Compound A treatment, associated with an increase in ejection fraction from 29% to 49%. This same improvement in ejection fraction also correlated with increased cardiac tissue spatial coverage of the human BAG3 transgene as determined using in situ hybridization (human BAG3 mRNA; 52% cardiomyocyte hBAG3 positive at 9E13vg / kg) and immunohistochemistry (BAG3 protein; 52% cardiac area hBAG3 positive at 9E13vg / kg). All measures of protein and mRNA expression also correlated with each other across dose groups.

[0390] Notably, the relationship between human BAG3 mRNA / protein expression and improvement in cardiac function tended to saturate above approximately 20% wild-type mouse cardiac BAG3 protein expression and approximately 20% human BAG3 mRNA+ cardiomyocytes, as determined by ISH (Figures 13A-B). Comparison of efficacy in individual mice separated by hBAG3 protein or ISH expression showed that mice with expression levels >20% by either measure had better ejection fraction changes than mice with <20% expression, but were comparable to mice with >40% expression (Figures 13C-D). These results suggest that improvement in cardiac function, as measured by LC / MS-based bulk protein levels and ISH-based cardiomyocyte coverage, saturates around approximately 20% BAG3 expression; therefore, this expression level was selected as the target therapeutic level of BAG3 expression.

[0391] Administration to humans To determine the human dose required to achieve target therapeutic levels of BAG3 expression, data from a dose-response study of Compound A in cynomolgus monkeys was used in conjunction with a developed quantitative systems pharmacology (QSP) model and validated using available literature and in-house data on interspecies transfer of AAV gene therapy.

[0392] BAG3 biodistribution was evaluated in cynomolgus monkeys (ETS) after IV administration of Compound A at 4E13 and 1.3E14 vg / kg (doses based on final viral titers determined post-study—updated from the original study design of 3E13 and 1E14 vg / kg). The observed cardiac transduction of Compound A, measured as viral genome copies per diploid genome equivalent (VCN / dge), increased in a dose-linear manner in cynomolgus monkeys (Figure 14A). The observed transduction was consistent with the model-predicted cardiac transduction for AAV9 as well as previous literature and in-house data for this capsid. This enabled us to use this model to predict cardiac transduction for Compound A in humans. Note that model predictions and available clinical data for AAV9 gene therapy indicated that the cardiac transduction dose-dependence for Compound A can be expected to be very similar between non-human primates and humans.

[0393] The observed cardiac cardiomyocyte coverage of Compound A, measured using ISH for hBAG3 mRNA, also increased in a dose-dependent manner in cynomolgus monkeys (FIG. 14B). Cardiac tissue coverage exceeded the target level of >20% cardiomyocytes at both the 4E13 and 1.3E14 vg / kg doses in monkeys and was expected to be translatable between monkeys and humans. Therefore, any Compound A dose above 4E13 vg / kg was expected to be sufficient to achieve this therapeutic target level.

[0394] A dose-dependent increase in hBAG3 protein levels (measured using LC / MS) in cynomolgus monkey hearts was also observed with Compound A (Figure 14C). Although data were only available for one monkey at the high dose, the increase in protein levels was greater than dose-linear (a 7-fold increase from 4E13 to 1.3E14 vg / kg). This data was modeled using a power law and included in a QSP model, allowing the dose-dependent BAG3 protein expression of Compound A in monkeys to be predicted, as shown in Figure 14C.

[0395] To extend predictions to humans, endogenous BAG3 protein expression was measured in untreated healthy cynomolgus monkeys and healthy human heart samples. These measurements showed approximately 1.7-fold lower BAG3 protein levels in human hearts compared to monkey hearts (human: 110 ng BAG3 / mg protein vs. cynomolgus monkey: 190 ng BAG3 / mg protein). Therefore, a similar 1.7-fold lower protein expression efficiency was assumed for the human protein projection model. Based on these assumptions, the model-predicted BAG3 protein dose dependence in humans is shown in Figure 14C. Using this predicted dose dependence, the clinical dose required to achieve the target level of BAG3 protein expression in human hearts (22 ng BAG3 / mg protein, based on 20% of the 110 ng BAG3 / mg protein in normal healthy human hearts) was predicted to be approximately 1E14 vg / kg of Compound A delivered intravenously. Note that if the 1.7x monkey-to-human correction factor is not used, and instead human protein expression is assumed to follow the cynomolgus dose-dependence, the projected clinical dose is approximately 7E13 vg / kg of Compound A delivered intravenously.

[0396] However, even less than maximal efficacy may be beneficial to patients. For example, a 5% difference in ejection fraction from placebo is measurable and clinically meaningful. At 3E13vg / kg, the change in ejection fraction is estimated to be approximately 5-10%. Therefore, an estimated dose of approximately 3E13vg / kg may define the lower limit of the clinical dose range.

[0397] In summary, approximately 3E13-1E14 vg / kg of Compound A is predicted to be the minimal effective dose range expected to achieve the target levels of BAG3 protein expression and cardiomyocyte coverage required for therapeutic benefit in patients. There are three main sources of uncertainty in this dose projection.

[0398] In some embodiments, a pharmaceutically effective amount of the rAAV vector ranges from about 1E10 to about 1E17 vector genomes per kilogram of subject body weight (vg / kg).

[0399] Example 9: In vivo efficacy study of Compound A in a BAG3 cardiac heterozygous mouse model The primary in vivo pharmacology of Compound A was investigated in mouse models of the disease to assess efficacy in relation to dose response. Mouse models of the disease were generated by cardiac-specific knockout of one or both BAG3 alleles, resulting in partial (BAG3 cHET mice) or complete (BAG3 cKO mice) knockdown of BAG3 expression in the heart. These mice have partial BAG3 expression in cardiomyocytes (approximately 50%, BAG3 cHET) or a complete lack of BAG3 expression (BAG3 cKO) and develop cardiac dysfunction and dilation, cardiac fibrosis, and express biomarkers of heart failure and cardiac fibrosis (Fang et al., J. Clin. Invest. 127(8):3189-200 (2017)). BAG3 cKO mice develop disease phenotypes at a faster rate than BAG3 cHET mice.

[0400] To determine whether restoring BAG3 expression in the heart via AAV gene delivery would result in improved cardiac structure and function or improved survival, a dose-response efficacy study was conducted in the BAG3 cHET mouse model of disease. BAG3 cHET mice were administered Compound A at 3E13vg / kg and 1E14vg / kg. Cardiac structure and function, as well as survival, were assessed longitudinally. At necropsy, AAV biodistribution and transgene expression (mRNA, protein) were assessed in the heart. HSPB8 stabilization (a surrogate for BAG3 expression), as well as biomarkers of heart failure (Nppa, Nppb, Myh6, Myh7), and apoptosis (Col1a1, Col1a2, Postn, Fn1, Timp1) were also assessed in the heart.

[0401] Methods for dose-response efficacy and survival studies BAG3 cHET mice aged 27–28 weeks were intravenously administered Compound A at 3E13vg / kg (n:14) and 1E14vg / kg (n:14). The study also included untreated control cohorts of BAG3 cHET mice (n:14) and BAG3 cWT mice (n:15). Cardiac structure and function were assessed using echocardiography at 25–26 weeks (baseline, 2 weeks before treatment), as well as at 30–31 weeks (3 weeks after treatment), 35–36 weeks (8 weeks after treatment), and 40–41 weeks (13 weeks after treatment). Survival was monitored for approximately 14 weeks after treatment.

[0402] At necropsy, cardiac tissue was collected for molecular biology analysis: AAV biodistribution and transgene expression (mRNA, protein). Prior to treatment, mice were randomly divided into groups based on echocardiographic readings (ejection fraction, left ventricular end-diastolic volume), body weight, and sex.

[0403] Echocardiographic evaluation For echocardiographic imaging, mice were induced to anesthesia using isoflurane (3%) or sevoflurane (5–6%) in an induction chamber, and anesthesia was then maintained at approximately 2% isoflurane or approximately 4–4.5% sevoflurane during animal preparation and image acquisition. While anesthetized, the animals were transferred to a water-circulating heating blanket, and hair around the left and ventral chest regions was removed using a chemical depilatory. After hair removal, each animal was transferred to a heated (approximately 34–35°C) platform for echocardiography. Cardiac transthoracic parasternal long-axis B-mode and parasternal short-axis M-mode images were acquired using an 18–38 MHz transducer (VisualSonics MS400®) on a Vevo 2100® or Vevo 3100® ultrasound system to assess left ventricular (LV) structure and function. The anesthesia level was adjusted to maintain a heart rate of 450–550 bpm during image acquisition. After image acquisition, mice were transferred to a warm cage for recovery. After image acquisition, images were analyzed using Vevo lab® analysis software or an automated analysis tool. The following parameters were collected from image analysis: LV ejection fraction, fractional shortening, LV end-diastolic and end-systolic volumes, LV end-diastolic and end-systolic areas, and LV posterior and anterior walls.

[0404] Survival Monitoring Assessment The animals were monitored daily for general health. Additionally, they were monitored once or twice weekly for general signs and symptoms of heart failure, including abnormal breathing, poor body condition (<2 of 5 animals), and poor activity / cage movement. Echocardiograms were performed every 3 to 5 weeks to assess cardiac function and structure. While being monitored for signs of heart failure, all animals were scored using an in-house heart failure scoring chart to grade the level of disease burden.

[0405] [Table 12]

[0406] tissue analysis Frozen tissue samples were used for DNA isolation using the phenol / chloroform DNA extraction method. The DNA pellet was washed with ice-cold 70% ethanol and resuspended in nuclease-free water. DNA concentration was measured using a Qiaxpert.

[0407] RNA isolation from frozen tissue was prepared by homogenizing approximately 20 mg of tissue sample in 1 mL of Trizol in the presence of 5 mm stainless steel beads for 5 minutes using a Tissuelyzer II set at 25 Hz. The lysate was transferred to a phasemaker tube, and 200 mL of chloroform was added. The sample was vortexed for 15 seconds, incubated at room temperature for 5 minutes, and centrifuged at 14,000 g for 5 minutes at 4°C. The clear supernatant was transferred to a new tube and treated with the RNeasy RNA Mini Kit according to the manufacturer's instructions. DNase treatment was performed on the column for 20 minutes before elution with nuclease-free water. RNA concentration was measured using a Nanodrop spectrophotometer.

[0408] Protein isolated from frozen tissue was prepared by homogenizing approximately 20 mg of tissue sample in 100 μL of RIPA buffer containing 1× protease inhibitors for 5 min in the presence of 5 mm stainless steel beads using a Tissuelyzer II set at 25 Hz. The lysate was incubated on wet ice for 30 min and centrifuged at maximum speed for 30 min at 4°C. The clear supernatant was transferred to a new tube. Protein concentration was measured using a BCA assay kit.

[0409] Vector Genome Quantification (VGC) VGC quantification was performed using ddPCR using primer and probe sequences to amplify the BAG3 transgene in all samples. The forward primer sequence was GGCTGGCCCTTCTTCGT (SEQ ID NO: 16). The reverse primer sequence was GCCCTCAGAAGGCACTCT (SEQ ID NO: 17). The probe sequence was CCACAATAGCAGAACCAC (SEQ ID NO: 18).

[0410] Quantification of transgene mRNA (BAG3) expression The isolated RNA samples were reverse transcribed to generate cDNA libraries using the SuperScript IV First-Stand Synthesis System according to the manufacturer's protocol. 100 or 400 ng of total RNA was used in a 20 μL reaction. Reverse transcription was performed at 23°C for 10 minutes, 55°C for 10 minutes, and then the reaction was inactivated at 80°C for 10 minutes and 5 minutes. The cDNA samples were stored at -20°C until RT-PCR analysis.

[0411] The cDNA was diluted 1:10 or 1:40 using nuclease-free HO to produce a final RNA amount of 2.5 ng in each ddPCR reaction. Quantification of BAG3 transgene mRNA expression was determined using ddPCR with the same BAG3 primers and probes as described in the previous section, as well as a housekeeping gene mTBP primer probe. PCR reaction mixtures containing ddPCR Supermix (without dUTP) and the above primer probes were prepared in an Airclean 600 PCR workstation and added to a ddPCR 96-well plate. Droplet generation, PCR programming, and data acquisition were performed as described in the previous section on VGC evaluation. mRNA expression was calculated by the ratio of the FAM to VIC channels.

[0412] Assessment of total BAG3 / HSPB8 protein expression Protein expression of total BAG3 (human and mouse), human BAG3, and HSPB8 was detected using Wes / Jess from ProteinSimple under optimized conditions (protein amount and antibody dilution) according to the manufacturer's instructions. GAPDH was detected as a loading control. The chemiluminescence intensity of each detected protein was automatically generated by the instrument software. Protein expression of total BAG3, human BAG3, and HSPB8 was quantified by the ratio of each protein to GAPDH.

[0413] Quantification of biomarkers of heart failure and fibrosis A total of 400 ng of RNA was used to prepare cDNA using the SuperScript IV Vilo RT kit with ezDNase, according to the manufacturer's instructions, resulting in a final cDNA concentration of 20 ng / μL. For each RT reaction and no-RT control reaction, 10 μL of gDNA reaction mixture was prepared by combining 1 μL of 10x ezDNase buffer, 1 μL of ezDNase enzyme, RNA (400 ng), and nuclease-free water. The samples were incubated at 37°C for 2 minutes and returned to ice. RT and no-RT reactions were then prepared. For each RT reaction, 4 μL of SSIV Vilo Master Mix was combined with 6 μL of nuclease-free water, and for each no-RT control reaction, 4 μL of SSIV Vilo No-RT Control Master Mix was combined with 6 μL of nuclease-free water. 10 μL of the master mix (RT or no-RT, respectively) was mixed with 10 μL of the gRNA reaction to bring the reaction to a final volume of 20 μL. RT was performed for 10 min at 25° C., 10 min at 50° C., and 5 min at 85° C. Samples were stored at −20° C. until RT-qPCR analysis.

[0414] cDNA was prepared in a 96-well PCR plate. A qPCR master mix was then prepared with the cDNA template. The cDNA was diluted 1:10 or 1:40 using nuclease-free HO to produce a final RNA amount of 2.5 ng in each ddPCR reaction. For each sample, the following was mixed: 55 μL of TaqMan Fast Advanced Master Mix (2x), 2.5 μL of 20 ng / μL cDNA, and 52.5 μL of nuclease-free water. 100 μL of the prepared PCR mix was then loaded into each reservoir on the array card. A total of seven samples and one no-reverse transcription (RT) control were added to each plate (a total of eight reservoirs / plate). The array card was centrifuged to ensure all liquid was at the bottom of the tube. The array card was removed from the centrifuge and sealed using a TaqMan Array Card Sealer. After sealing, the filled reservoir strips were cut from the card using scissors. The array cards were run on a ViiA 7 qPCR instrument using the standard cycling protocol for the array cards and TaqMan Fast Advanced Master Mix (50°C for 2 minutes, 95°C for 20 seconds, [95°C for 1 second, 60°C for 20 seconds] x 40). Results were analyzed using relative quantification to the housekeeping gene HPRT.

[0415] [Table 13]

[0416] Results and Discussion BAG3 cHET mice (mixed sex) were treated (age: 27-28 weeks) with 3E13vg / Kg and 1E14vg / Kg of Compound A. The study also included untreated control cohorts of cHET mice and cWT mice.

[0417] Cardiac function (ejection fraction) and structure (left ventricular end-diastolic and end-systolic volumes) were assessed longitudinally using echocardiography at 25–26 weeks of age (baseline, 2 weeks before treatment), 30–31 weeks of age (3 weeks after treatment), 35–36 weeks of age (8 weeks after treatment), and 40–41 weeks of age (13 weeks after treatment) (Figures 15A–15C). When measuring cardiac ejection fraction at all three post-treatment time points, no significant differences were detected when comparing treated mice (3E13vg / kg or 1E14vg / kg) with untreated BAG3 cHET control cohorts. Significant decreases were detected within all cHET groups when comparing values ​​collected at baseline and 8 or 13 weeks after treatment. When LVEDV was measured at all three time points after treatment, no significant differences were detected when comparing treated mice (3E13vg / Kg or 1E14vg / Kg) with the untreated BAG3 cHET control cohort. A significant increase was detected in the untreated control group when comparing values ​​collected at baseline and 8 or 13 weeks after treatment. When LVESV was measured at all three time points after treatment, no significant differences were detected when comparing treated mice (3E13vg / Kg or 1E14vg / Kg) with the untreated BAG3 cHET control cohort. A significant increase was detected in the untreated BAG3 cHET control group when comparing values ​​collected at baseline and 8 or 13 weeks after treatment.

[0418] Survival was assessed longitudinally up to 42 weeks (approximately 14 weeks post-treatment) based on the predefined set of criteria described above. No significant differences in survival were detected when comparing treated mice (3E13vg / Kg or 1E14vg / Kg) with untreated BAG3 cHET control cohorts (Figure 16A). No significant differences in body weight were detected between these mouse groups (Figure 16B).

[0419] Viral genome biodistribution was investigated in cardiac tissue collected at autopsy from BAG3 cHET mice treated with 3E13vg / kg and 1E14vg / kg of Compound A (Figure 17A). A dose response in biodistribution was detected in the heart: 3E13vg / kg (0.131±0.066vg / mTFRC), 1E14vg / kg (0.371±0.107vg / mTFRC).

[0420] Transgene mRNA expression was investigated in cardiac tissue collected at autopsy from mice treated with 3E13vg / kg and 1E14vg / kg of Compound A (FIG. 17B). A dose response in expression was detected in the heart: 3E13vg / kg (10.81±6.18vg / mTBP), 1E14vg / kg (50.889±14.607vg / mTBP).

[0421] BAG3 protein expression was examined in cardiac tissue collected at necropsy from mice treated with Compound A at 3E13vg / kg and 1E14vg / kg (Figure 17C). Protein expression was normalized to the level of BAG3 expression detected in the hearts of a cohort of wild-type mice. A dose-responsive increase in BAG3 protein levels was observed in BAG3 cHET mouse hearts after treatment with Compound A. At the high dose level (1E14vg / Kg), approximately 90% of the WT level of BAG3 (0.912±0.196) was detected, and at the low dose (3E13vg / Kg), approximately 60% of the WT BAG3 level (0.607±0.134) was observed. Approximately 40% of the WT level of BAG3 (0.392±0.096) was detected in untreated cHET BAG3 mice.

[0422] HSPB8 protein expression was examined in cardiac tissue collected at autopsy from mice treated with Compound A at 3E13vg / kg and 1E14vg / kg (FIG. 17D). Protein expression was normalized to the level of BAG3 expression detected in the hearts of a cohort of wild-type mice. A dose-responsive increase in HSPB8 protein levels was observed in the hearts of BAG3 cHET mice after treatment with Compound A. At the high dose level (1E14vg / Kg), approximately 85% (0.855±0.155) of the WT level of BAG3 was detected, and at the low dose (3E13vg / Kg), approximately 63% (0.637±0.117) of the WT BAG3 level was observed. Approximately 46% (0.460±0.071) of the WT level of BAG3 was detected in untreated cHET BAG3 mice.

[0423] Biomarkers of fibrosis (Col1a1, Col1a2, Fn1, Postn, Timp1) and heart failure (Nppa, Nppb, Myh7, Myh6) were assessed (RNA expression) in the hearts of treated and untreated mice. When measuring biomarkers of fibrosis, no significant differences were detected when comparing treated mice (3E13vg / kg or 1E14vg / kg) with untreated BAG3 cHET control cohorts (Figures 18A-E). When measuring biomarkers of heart failure, no significant differences were detected when comparing treated mice (3E13vg / kg or 1E14vg / kg) with untreated BAG3 cHET control cohorts (Figures 18F-H).

[0424] conclusion At 27-28 weeks, treatment of BAG3 cHET mice with Compound A did not result in improved cardiac structure, cardiac function, or survival compared with untreated control BAG3 cHET mice. A dose-dependent increase in viral genome expression in the heart correlated with a dose-dependent increase in transgene mRNA and protein expression and restoration of HSPB8 protein expression. No significant improvements were detected in biomarkers of heart failure or fibrosis.

[0425] Example 10: Survival and efficacy studies of Compound A in a BAG3 in vivo cardiac knockout mouse model The primary pharmacology of Compound A in vivo was investigated in wild-type mice to assess efficacy in relation to dose response. A mouse model of the disease was generated by cardiac-specific knockout of BAG3 expression (BAG3 cKO mice). These mice lack BAG3 expression in cardiomyocytes and develop cardiac dysfunction and dilation, cardiac fibrosis, and express biomarkers of heart failure and cardiac fibrosis (Fang et al., J. Clin. Invest. 127(8):3189-200(2017)).

[0426] To determine whether restoring BAG3 expression in the heart via AAV gene delivery results in improved survival, a dose-response efficacy study was conducted in a cKO mouse model of disease. BAG3 cKO mice were administered 3E13vg / kg and 1E14vg / kg of Compound A. Survival, cardiac structure, and function were assessed longitudinally using echocardiography. At necropsy, AAV biodistribution and transgene expression (mRNA, protein) were assessed in the heart.

[0427] Methods for dose-response survival studies BAG3 cKO mice aged 14–15 weeks were intravenously administered Compound A at 3E13vg / kg (n:16) and 1E14vg / kg (n:15). The study also included a control cohort of vehicle-treated cKO mice (n:13). Survival was monitored for approximately 21 weeks after treatment. Cardiac structure and function were assessed using echocardiography at baseline (1 week before treatment) and at 2, 5, 9, and 13 weeks after treatment. At necropsy, cardiac tissue was collected for molecular biology analysis: AAV biodistribution, and transgene expression (mRNA, protein). Prior to treatment, mice were randomly divided into groups based on echocardiographic readings (ejection fraction, left ventricular end-diastolic volume), body weight, and sex.

[0428] General method The following methods were performed as described in Example 9: DNA isolation, RNA isolation, protein preparation, VGC quantification, quantification of BAG3 transgene RNA expression, quantification of total BAG3 protein expression, echocardiographic evaluation, and survival monitoring evaluation.

[0429] Results and Discussion BAG3 cKO mice (mixed sex) were treated with vehicle or either 3E13vg / kg or 1E14vg / kg of Compound A (age: 14-15 weeks). Survival (Figure 19A) was assessed longitudinally (up to approximately 16 weeks post-treatment) based on a set of predefined criteria (see Example 9). Vehicle-treated mice (Group 1) were determined to have a significantly lower survival rate than mice treated with Compound A at either 3E13vg / kg (Group 2) or 1E14vg / kg (Group 3). No significant differences in body weight were detected in these mouse groups (Figure 19B).

[0430] Cardiac function (ejection fraction) and structure (left ventricular end-diastolic and end-systolic volumes) were assessed longitudinally using echocardiography at baseline and 2, 5, 9, and 13 weeks after treatment (Figures 20A-C). At 13 weeks after treatment, significant improvements in ejection fraction were detected in mice treated with Compound A at 3E13vg / kg (41.0±17.1%) and 1E14vg / kg (51.2±16.6%) compared to vehicle-treated BAG3 cKO mice (27.0±15.8%). At 13 weeks, significant improvements in left ventricular end-diastolic and end-systolic volumes were also detected in mice treated with 1E14vg / kg Compound A (LVEDV: 54.7±23.7uL, LVESV: 29.8±26.5uL) compared to vehicle-treated BAG3 cKO mice (LVEDV: 75.6±25.1uL, LVESV: 57.6±28.3uL).

[0431] The biodistribution of viral genome was investigated in cardiac tissue collected at autopsy from mice treated with 3E13vg / kg and 1E14vg / kg of Compound A (Figure 21A). A dose response in biodistribution was detected in the heart: 3E13vg / kg (0.080±0.039vg / mTFRC), 1E14vg / kg (0.32±0.13vg / mTFRC).

[0432] Transgene mRNA expression was investigated in cardiac tissue collected at autopsy from mice treated with 3E13vg / kg and 1E14vg / kg of Compound A (Figure 21B). A dose response in expression was detected in the heart: 3E13vg / kg (8.21±5.59vg / mTFRC), 1E14vg / kg (43.91±15.99vg / mTFRC).

[0433] BAG3 protein expression was also examined in cardiac tissue collected at necropsy from mice treated with 3E13vg / kg and 1E14vg / kg Compound A (Figure 21C). Protein expression was normalized to the level of BAG3 expression detected in the hearts of a cohort of wild-type mice. A dose-responsive increase in BAG3 protein levels was observed in cKO mouse hearts after treatment with Compound A. At the high dose level (1E14vg / Kg), approximately 50% (0.49±0.16) of WT levels of BAG3 were detected, and at the low dose (3E13vg / Kg), approximately 8% (0.08±0.04) of WT BAG3 levels were observed.

[0434] conclusion Treatment of BAG3 cKO mice with Compound A resulted in improved survival and dose-dependent improvements in both cardiac structure and function, which correlated with the expression of human BAG3 protein in the heart.

[0435] Example 11: Clinical Trials BAG3 is involved in diverse cellular functions, including but not limited to excitation-contraction coupling, maintenance of sarcomere integrity, and regulation of autophagy. Comprehensive assessment of disease manifestations caused by BAG3 mutations in the general population is complicated by the rarity of the disease and the single-center nature of most case reports of BAG3-related disease. The precise characterization of BAG3 DCM using imaging and circulating biomarkers at symptom onset, as well as its clinical progression, remains unclear. The development of novel transformative therapies for BAG3 DCM requires a clear understanding of circulating, imaging, and clinical biomarkers at baseline and over time. Therefore, longitudinal natural history studies are being conducted in patients diagnosed with dilated cardiomyopathy caused by BAG3 mutations that are likely pathogenic or pathological.

[0436] Eligible subjects from this longitudinal study may be enrolled in a two-part, Phase 1b, interventional, open-label, dose-escalation study followed by a Phase 2, placebo-controlled, double-blind study to investigate the safety, tolerability, and efficacy of a single dose of ALXN2350 in participants with dilated cardiomyopathy associated with BAG3 mutations.

[0437] This prospective, open-label, dose-evaluation Phase 1b followed by a blinded, placebo-controlled Phase 2 study is a first-in-human (FIH) / first-in-patient (FIP) trial using adeno-associated virus, serotype 9 (AAV9)-based gene therapy compound A. The study aims to evaluate the safety, tolerability, and efficacy of a single dose of compound A in participants with dilated cardiomyopathy caused by likely pathogenic or pathogenic BAG3 mutations. Other objectives include exploratory assessments related to long-term safety, dose selection, quality of life, and functional capacity to support future clinical development.

[0438] A two-part design is proposed, with sequential implementation of Phase 1b and Phase 2, with each part conducted in a separate / distinct cohort of participants. Phase 1b of the study aims to explore safety, tolerability, and immunogenicity across a dose range from a planned starting minimum active dose of approximately 3E13vg / kg to a maximum feasible dose of approximately 1E14vg / kg. To enable blinded assessment of efficacy, Phase 2 of the study will compare the proposed clinical dose levels discovered in Phase 1b with a matching placebo in a randomized, double-blind design. After the initial 12 months of efficacy and safety follow-up, all eligible participants in the Phase 2 portion who received placebo will be offered the opportunity to cross over to Compound A intervention to enable an extension of the safety and efficacy investigation (using a delayed-start analysis to further evaluate the impact of therapy on disease progression). The double-blind nature of the study will be maintained until all participants have completed at least 12 months of follow-up and an independent data monitoring committee determines eligibility for an open-label extension.

[0439] The Phase 1b / 2 trial will be followed by a Phase 3 double-blind, randomized, placebo-controlled trial with primary efficacy endpoints of functional capacity and symptom improvement. This pivotal Phase 3 registrational trial will undergo an interim analysis to examine the effect of the investigational drug on positive cardiac remodeling. This interim analysis will utilize both futility and overwhelming success criteria. If the level of positive (reversed) remodeling achieves a predetermined success level compared with placebo, this data will be utilized for an early accelerated approval strategy.

[0440] [Table 14]

[0441] [Table 15]

[0442] Selecting a target Subjects included in the protocol must meet specific inclusion criteria, including but not limited to: male / female; known BAG-3 mutation (likely pathological / pathological), age ≥ 18 years; LVEF < 50%; stage B (NYHA I / IV) subjects will be excluded if the subject has CKD ≥ IIIb; non-BAG3-related heart disease; transaminases > 2 × ULN; T.Bili > 1.5 × ULN.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof.

2. 2. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is a codon-optimized nucleotide sequence.

3. 3. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

4. 4. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

5. 5. The nucleic acid molecule of any one of claims 1 to 4, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

6. 6. The nucleic acid molecule of any one of claims 1 to 5, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

7. 7. The nucleic acid molecule of any one of claims 1 to 6, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

8. 8. The nucleic acid molecule of any one of claims 1 to 7, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

9. 9. The nucleic acid molecule of any one of claims 1 to 8, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

10. The nucleic acid molecule according to any one of claims 1 to 9, wherein the nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

11. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid molecule of any one of claims 1 to 10.

12. 12. The rAAV vector of claim 11, wherein the rAAV vector comprises a serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

13. The rAAV vector of claim 11 or 12, wherein the rAAV vector comprises an AAV9 capsid protein.

14. 14. The rAAV vector of claim 13, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof; a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof; or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

15. 15. The rAAV vector of claim 13 or 14, wherein the AAV9 capsid proteins include the VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof, the VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof, and the VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

16. The rAAV vector of any one of claims 11 to 15, wherein the rAAV vector further comprises at least one cardiac promoter operably linked to the nucleic acid molecule comprising the nucleotide sequence encoding a BAG3 polypeptide or a variant thereof.

17. 17. The rAAV vector of claim 16, wherein the at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant thereof.

18. 18. The rAAV vector of claim 16 or 17, wherein the at least one cardiac promoter is a cTNT promoter, or a functional subsequence, modification, or variant thereof.

19. 19. The rAAV vector of claim 17 or 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

20. 19. The rAAV vector of claim 17 or 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO:

5.

21. The rAAV vector of any one of claims 11 to 20, wherein the rAAV vector further comprises at least one intron.

22. 22. The rAAV vector of claim 21, wherein the intron comprises the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, or a functional subsequence, modification, or variant thereof.

23. 23. The rAAV vector of claim 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

6.

24. 23. The rAAV vector of claim 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

29.

25. 23. The rAAV vector of claim 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

30.

26. 23. The rAAV vector of claim 21 or 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

31.

27. The rAAV vector of any one of claims 11 to 26, wherein the rAAV vector further comprises at least one 5' inverted terminal repeat (ITR) sequence.

28. 28. The rAAV vector of claim 27, wherein the at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

29. 29. The rAAV vector of claim 27 or 28, wherein the at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO:

8.

30. The rAAV vector of any one of claims 11 to 29, wherein the rAAV vector further comprises at least one 3' ITR sequence.

31. 31. The rAAV vector of claim 30, wherein the at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

32. 32. The rAAV vector of claim 30 or 31, wherein the at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:

9.

33. The rAAV vector of any one of claims 11 to 32, wherein the rAAV vector further comprises at least one transcription termination sequence.

34. 34. The rAAV vector of claim 33, wherein the at least one transcription termination sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

35. 35. The rAAV vector of claim 33 or 34, wherein the at least one transcription termination sequence is an SV40 polyA sequence.

36. 36. The rAAV vector of any one of claims 33 to 35, wherein the at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

37. The rAAV vector of any one of claims 33 to 36, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO:

7.

38. 38. The rAAV vector of any one of claims 11 to 37, wherein the rAAV vector further comprises at least one stuffer or filler sequence, preferably the at least one stuffer or filler sequence including the ITR sequences, increasing the overall length of the nucleic acid molecule of the rAAV vector to approximately 4.2 to 4.7 kilobases.

39. 39. The rAAV vector of claim 38, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11, or a functional subsequence, modification, or variant thereof.

40. 40. The rAAV vector of claim 38 or 39, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO:

11.

41. A rAAV vector comprising, in 5' to 3' order: (a) at least one 5' ITR sequence; (b) at least one cardiac promoter; (c) at least one intron; and (d) at least one nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof, operably linked to the at least one cardiac promoter; (e) at least one transcription termination sequence; (f) at least one stuffer or filler sequence; (g) at least one 3' ITR sequence.

42. 42. The rAAV vector of claim 41, wherein the rAAV vector comprises a serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

43. 43. The rAAV vector of claim 41 or 42, wherein the rAAV vector comprises an AAV9 capsid protein.

44. 44. The rAAV vector of claim 43, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof; a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof; or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

45. 45. The rAAV vector of claim 43 or 44, wherein the AAV9 capsid proteins include the VP1 protein comprising the amino acid sequence of SEQ ID NO: 1, or a functional subsequence, modification, or variant thereof, the VP2 protein comprising the amino acid sequence of SEQ ID NO: 2, or a functional subsequence, modification, or variant thereof, and the VP3 protein comprising the amino acid sequence of SEQ ID NO: 3, or a functional subsequence, modification, or variant thereof.

46. 1. An rAAV vector plasmid comprising, in 5' to 3' order: (a) at least one left spacer sequence; (b) at least one 5' ITR sequence; (c) at least one cardiac promoter; (d) at least one intron; and (e) at least one nucleotide sequence encoding a Bcl2-associated athanogen 3 (BAG3) polypeptide, or a variant thereof, operably linked to the at least one cardiac promoter; (f) at least one transcription termination sequence; (g) at least one stuffer or filler sequence; (h) at least one 3' ITR sequence; (i) at least one right spacer sequence.

47. 47. The rAAV vector of any one of claims 41 to 45, or the rAAV vector plasmid of claim 46, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is a codon-optimized nucleotide sequence.

48. 48. The rAAV vector of any one of claims 41 to 45 or 47, or the rAAV vector plasmid of claim 46 or 47, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

49. 49. The rAAV vector of any one of claims 41 to 45, 47, or 48, or the rAAV vector plasmid of any one of claims 46 to 48, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

50. 50. The rAAV vector of any one of claims 41 to 45 or 47 to 49, or the rAAV vector plasmid of any one of claims 46 to 49, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

51. 51. The rAAV vector of any one of claims 41 to 45 or 47 to 50, or the rAAV vector plasmid of any one of claims 46 to 50, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

52. 52. The rAAV vector of any one of claims 41 to 45 or 47 to 51, or the rAAV vector plasmid of any one of claims 46 to 51, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

53. 53. The rAAV vector of any one of claims 41 to 45 or 47 to 52, or the rAAV vector plasmid of any one of claims 46 to 52, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

54. 54. The rAAV vector of any one of claims 41 to 45 or 47 to 53, or the rAAV vector plasmid of any one of claims 46 to 53, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or a variant thereof, is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

55. 55. The rAAV vector of any one of claims 41 to 45 or 47 to 54, or the rAAV vector plasmid of any one of claims 46 to 54, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide or a variant thereof is the nucleotide sequence of SEQ ID NO: 4 or 19 to 24.

56. 56. The rAAV vector of any one of claims 41 to 45 or 47 to 55, or the rAAV vector plasmid of any one of claims 46 to 55, wherein the at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

57. 57. The rAAV vector of any one of claims 41 to 45 or 47 to 56, or the rAAV vector plasmid of any one of claims 46 to 56, wherein the at least one 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO:

8.

58. 58. The rAAV vector of any one of claims 41-45 or 47-57, or the rAAV vector plasmid of any one of claims 46-57, wherein the at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant thereof.

59. 59. The rAAV vector or rAAV vector plasmid of claim 58, wherein the at least one cardiac promoter is a cTNT promoter, or a functional subsequence, modification, or variant thereof.

60. 60. The rAAV vector or rAAV vector plasmid of claim 58 or 59, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

61. 61. The rAAV vector or rAAV vector plasmid of any one of claims 58 to 60, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO:

5.

62. 62. The rAAV vector of any one of claims 41-45 or 47-61, or the rAAV vector plasmid of any one of claims 46-61, wherein the at least one intron comprises the nucleotide sequence of SEQ ID NO:6, or a functional subsequence, modification, or variant thereof.

63. 63. The rAAV vector of any one of claims 41 to 45 or 47 to 62, or the rAAV vector plasmid of any one of claims 46 to 62, wherein the at least one intron comprises the nucleotide sequence of SEQ ID NO:

6.

64. 64. The rAAV vector of any one of claims 41 to 45 or 47 to 63, or the rAAV vector plasmid of any one of claims 46 to 63, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

29.

65. 65. The rAAV vector of any one of claims 41 to 45 or 47 to 64, or the rAAV vector plasmid of any one of claims 46 to 64, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

30.

66. 66. The rAAV vector of any one of claims 41 to 45 or 47 to 65, or the rAAV vector plasmid of any one of claims 46 to 65, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

31.

67. 67. The rAAV vector of any one of claims 41 to 45 or 47 to 66, or the rAAV vector plasmid of any one of claims 46 to 66, wherein the at least one transcription termination sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

68. 68. The rAAV vector of any one of claims 41 to 45 or 47 to 67, or the rAAV vector plasmid of any one of claims 46 to 67, wherein the at least one transcription termination sequence is an SV40 polyA sequence.

69. 69. The rAAV vector of any one of claims 41-45 or 47-68, or the rAAV vector plasmid of any one of claims 46-68, wherein the at least one transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

70. 70. The rAAV vector of any one of claims 41 to 45 or 47 to 69, or the rAAV vector plasmid of any one of claims 46 to 69, wherein at least the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO:

7.

71. 71. The rAAV vector of any one of claims 41-45 or 47-70, or the rAAV vector plasmid of any one of claims 46-70, wherein the at least one stuffer or filler sequence increases the overall length of the rAAV vector or rAAV vector plasmid nucleic acid molecule, including the ITR sequences and excluding spacer sequences, to approximately 4.2 to 4.7 kilobases.

72. 72. The rAAV vector of any one of claims 41-45 or 47-71, or the rAAV vector plasmid of any one of claims 46-71, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11, or a functional subsequence, modification, or variant thereof.

73. 73. The rAAV vector of any one of claims 41-45 or 47-72, or the rAAV vector plasmid of any one of claims 46-72, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO:

11.

74. 74. The rAAV vector of any one of claims 41-45 or 47-73, or the rAAV vector plasmid of any one of claims 46-73, wherein the at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:9, or a functional subsequence, modification, or variant thereof.

75. 75. The rAAV vector of any one of claims 41 to 45 or 47 to 74, or the rAAV vector plasmid of any one of claims 46 to 74, wherein the at least one 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:

9.

76. 76. The rAAV vector plasmid of any one of claims 46-75, wherein the at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO: 12, or a functional subsequence, modification, or variant thereof.

77. 77. The rAAV vector plasmid of any one of claims 46 to 76, wherein the at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO:

12.

78. 78. The rAAV vector plasmid of any one of claims 46-77, wherein the at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO: 13, or a functional subsequence, modification, or variant thereof.

79. 79. The rAAV vector plasmid of any one of claims 46 to 78, wherein the at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO:

13.

80. 80. The rAAV vector plasmid of any one of claims 46 to 79, wherein the rAAV vector plasmid further comprises at least one left spacer and / or at least one right spacer, preferably wherein the at least one left spacer and / or at least one right spacer increases the overall length of the rAAV vector plasmid to approximately 4.7 to 12 kilobases, preferably approximately 9 to 10 kilobases.

81. 81. The rAAV vector of any one of claims 11 to 45 or 47 to 75, or the rAAV vector plasmid of any one of claims 46 to 80, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 14, or a functional subsequence, modification, or variant thereof.

82. 82. The rAAV vector of any one of claims 11 to 45, 47 to 75, or 81, or the rAAV vector plasmid of any one of claims 46 to 81, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO:

14.

83. 82. The rAAV vector of any one of claims 11 to 45, 47 to 75, 81, or 82, or the rAAV vector plasmid of any one of claims 46 to 82, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 15, or a functional subsequence, modification, or variant thereof.

84. The rAAV vector of any one of claims 11 to 45, 47 to 75, or 81 to 83, or the rAAV vector plasmid of any one of claims 46 to 83, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO:

15.

85. 85. The rAAV vector of any one of claims 11 to 45, 47 to 75, or 81 to 84, wherein the overall length of the rAAV vector nucleic acid molecule is approximately 5 kilobases or less in length, or 4.7 kilobases or less in length.

86. 85. The rAAV vector plasmid of any one of claims 46 to 84, wherein the overall length of the rAAV vector plasmid is approximately 12 kilobases or less in length, or 4.7 kilobases or less in length.

87. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 10, or the rAAV vector of any one of claims 11 to 45, 47 to 75, or 81 to 85, and at least one pharmaceutically acceptable salt.

88. 88. The pharmaceutical composition of claim 87, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 1 mM to about 450 mM.

89. 89. The pharmaceutical composition of claim 87 or 88, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 2 mM to about 350 mM.

90. 90. The pharmaceutical composition of any one of claims 87 to 89, wherein the at least one pharmaceutically acceptable salt is sodium chloride, magnesium chloride, potassium chloride, calcium chloride, or calcium phosphate.

91. 91. The pharmaceutical composition of claim 90, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and magnesium chloride.

92. 91. The pharmaceutical composition of claim 90, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and potassium chloride.

93. 93. The pharmaceutical composition of any one of claims 87 to 92, further comprising at least one buffering agent.

94. 94. The pharmaceutical composition of claim 93, wherein the buffering agent is citrate, histidine, acetate, phosphate, tris hydrochloride, or tromethamine.

95. 95. The pharmaceutical composition of claim 94, wherein the buffering agent is phosphate.

96. 95. The pharmaceutical composition of claim 94, wherein the buffering agent is tris hydrochloride and tromethamine.

97. 97. The pharmaceutical composition of any one of claims 93 to 96, wherein the at least one buffering agent is present in an amount ranging from about 10 mM to about 40 mM.

98. 98. The pharmaceutical composition of claim 97, wherein the at least one buffering agent is present in an amount of about 20 mM.

99. 99. The pharmaceutical composition of any one of claims 87 to 98, further comprising at least one cryoprotectant.

100. 100. The pharmaceutical composition of claim 99, wherein the at least one cryoprotectant is a sugar or sugar alcohol.

101. 100. The pharmaceutical composition of claim 99, wherein the at least one cryoprotectant is trehalose, sucrose, sorbitol, or mannitol.

102. 102. The pharmaceutical composition of claim 101, wherein the at least one cryoprotectant is sucrose.

103. 102. The pharmaceutical composition of claim 101, wherein the at least one cryoprotectant is sorbitol.

104. 104. The pharmaceutical composition of any one of claims 99-103, wherein the at least one cryoprotectant is present in an amount of up to about 20%.

105. 105. The pharmaceutical composition of claim 104, wherein the at least one cryoprotectant is present in an amount ranging from about 3% to about 15%.

106. 106. The pharmaceutical composition of claim 105, wherein the at least one cryoprotectant is present in an amount of about 4% or about 5%.

107. 107. The pharmaceutical composition of any one of claims 87 to 106, further comprising at least one surfactant.

108. 108. The pharmaceutical composition of claim 107, wherein the at least one surfactant is a polaxamer or a polysorbate.

109. 109. The pharmaceutical composition of claim 108, wherein the at least one surfactant is polaxamer 188, polysorbate 20, or polysorbate 80.

110. 110. The pharmaceutical composition of any one of claims 107 to 109, wherein the at least one surfactant is present in an amount ranging from about 0.0001% to about 1%.

111. 111. The pharmaceutical composition of claim 110, wherein the at least one surfactant is present in an amount of about 0.02%.

112. 111. The pharmaceutical composition of claim 110, wherein the at least one surfactant is present in an amount of about 0.002%.

113. 113. The pharmaceutical composition of any one of claims 87 to 112, wherein the pharmaceutical composition has a pH in the range of about 6 to about 8.

114. 114. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition has a pH in the range of about 7 to about 8.

115. 115. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition has a pH of about 7.

6.

116. 115. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition has a pH of about 7.

4.

117. 1. A method for treating a heart-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of (a) a nucleic acid molecule according to any one of claims 1 to 10, or (b) an rAAV vector according to any one of claims 11 to 45, 47 to 75, or 81 to 85; or (c) administering a pharmaceutical composition according to any one of claims 87 to 116.

118. 118. The method of claim 117, wherein the heart-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

119. 119. The method of claim 117 or 118, wherein the subject has a BAG3 mutation.

120. 120. The method of any one of claims 117 to 119, wherein the heart-related disease or disorder is BAG3-associated dilated cardiomyopathy (DCM).

121. 120. The method of any one of claims 117 to 119, wherein the heart-related disease or disorder is BAG3-associated heart failure.

122. 118. The method of claim 117, wherein the heart-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

123. 123. The method of claim 117 or 122, wherein the subject does not have a BAG mutation.

124. 124. The method of any one of claims 117, 122, or 123, wherein the heart-related disease or disorder is heart failure unrelated to BAG3 expression.

125. 1. A method for reducing the frequency or severity of at least one symptom associated with a heart-related disease or disorder in a subject, comprising administering to the subject: (a) a nucleic acid molecule according to any one of claims 1 to 10, or (b) an rAAV vector according to any one of claims 11 to 45, 47 to 75, or 81 to 85; or (c) a pharmaceutical composition according to any one of claims 87 to 116, administering in an amount effective to reduce the frequency or severity of said at least one symptom.

126. 126. The method of claim 125, wherein the heart-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

127. 127. The method of claim 125 or 126, wherein the subject has a BAG3 mutation.

128. 128. The method of any one of claims 125 to 127, wherein the heart-related disease or disorder is BAG3-associated dilated cardiomyopathy (DCM).

129. 128. The method of any one of claims 125 to 127, wherein the heart-related disease or disorder is BAG3-associated heart failure.

130. 126. The method of claim 125, wherein the heart-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

131. 131. The method of claim 125 or 130, wherein the subject does not have a BAG mutation.

132. 132. The method of any one of claims 125, 130, or 131, wherein the heart-related disease or disorder is heart failure unrelated to BAG3 expression.

133. 129. The method of any one of claims 125 to 128, wherein said at least one symptom is characteristic of BAG3-associated DCM.

134. 130. The method of any one of claims 125-127 or 129, wherein said at least one symptom is characteristic of BAG3-associated heart failure.

135. 133. The method of any one of claims 125 or 130 to 132, wherein said at least one symptom is characteristic of heart failure independent of BAG3 expression.

136. 136. The method of any one of claims 117-135, wherein the effective amount of the rAAV vector is in the range of about 1E10 to about 1E17 vector genomes per kilogram of subject body weight (vg / kg).

137. 137. The method of claim 136, wherein the effective amount of the rAAV vector is in the range of about 3E13 to about 1E14 vector genomes per kilogram of subject body weight (vg / kg).

138. 138. The method of claim 136 or 137, wherein the effective amount of the rAAV vector is about 3E13 vector genomes per kilogram of subject body weight (vg / kg).

139. 138. The method of claim 136 or 137, wherein the effective amount of the rAAV vector is about 7E13 vector genomes per kilogram of subject body weight (vg / kg).

140. 138. The method of claim 136 or 137, wherein the effective amount of the rAAV vector is 1E14 vector genomes per kilogram of subject body weight (vg / kg).

141. 11. Use of a nucleic acid molecule according to any one of claims 1 to 10 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

142. 100. Use of the rAAV vector of any one of claims 11-45, 47-75, or 81-85 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

143. 117. Use of the pharmaceutical composition of any one of claims 87 to 116 in the manufacture of a medicament for treating a heart-related disease or disorder in a subject.

144. A plasmid comprising the nucleic acid molecule of any one of claims 1 to 10.

145. A plasmid comprising the rAAV vector sequence of any one of claims 11 to 45, 47 to 75, or 81 to 85.

146. A host cell for producing an rAAV vector, comprising the plasmid of claim 144.

147. A host cell for producing an rAAV vector, comprising the plasmid of claim 145.

148. A host cell for producing an rAAV vector, comprising the rAAV vector plasmid of any one of claims 46 to 84.

149. The host cell of any one of claims 146 to 148, wherein the host cell is a HEK293 cell or a derivative thereof.

150. 150. The host cell of any one of claims 146 to 149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep protein.

151. 150. The host cell of any one of claims 146 to 149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV9 capsid protein.

152. 150. The host cell of any one of claims 146 to 149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a viral helper factor.

153. 1. A method for producing an rAAV vector, comprising: (a) incubating the host cell of any one of claims 146-152 under conditions sufficient to allow production of an rAAV vector; (b) purifying the rAAV vector produced thereby.

154. 153. An rAAV vector produced by the method of claim 152.

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